Therapeutic binders that conditionally promote myeloid cell activity against target cells and their use

Abstract

This disclosure provides, in particular, a therapeutic conjugate that can direct the activity of myeloid cells towards a target cell population (such as cancer cells).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 532,538, filed on 14 August 2023, the contents of which are incorporated in their entirety by reference.

[0002] Inclusion by referencing the sequence list This application is filed together with an electronic sequence listing. The sequence listing is provided as a 461,658-byte file titled 336402000140SeqList.XML, created on August 12, 2024. The electronic information of the sequence listing is incorporated in its entirety by reference. [Background technology]

[0003] background Myeloid cells, being immune cells found in virtually all tissues, contribute to immune responses and cell maintenance. In the tumor environment, myeloid cells are the most abundant immune cell type and exert diverse activities. Among the potential functions of myeloid cells (such as macrophages, granulocytes, monocytes, and dendritic cells (DCs)) is their ability to recognize cancer cells and / or contribute to antitumor responses, for example, by phagocytosis of cancer cells. [Overview of the project]

[0004] overview This disclosure provides therapeutic conjugates that can direct the activity of myeloid cells (such as direct death of undesirable cells (e.g., cancer cells or targeted immune cells), indirect death of undesirable cells (e.g., cancer cells or targeted immune cells), inhibition of target cells (e.g., targeted immune cells), and so on) to a target cell population. This disclosure provides therapeutic conjugates that can cause direct or indirect elimination of target cells by antibody-dependent cell-mediated cytotoxicity (ADCC) and / or by the activity of the therapeutic conjugate that directly kills target cells, for example, by directing the activity of myeloid cells (e.g., phagocytic activity of myeloid cells) to a target cell population. In various embodiments, the target cells may be, for example, cancer cells or targeted immune cells. This disclosure further provides that in some embodiments, such therapeutic conjugates selectively guide myeloid cells to target a cell population such as cancer cells (e.g., those in the tumor environment in a particular context). In some embodiments, the therapeutic conjugates encompassed by this disclosure lead to the elimination of target cells by directing the activity (e.g., phagocytic activity) of myeloid cells to a target cell population in a particular environment (e.g., cancer cells in a tumor environment, autoimmune cells in an inflammatory environment) through a combination of elements that may include, for example, (1) at least one binding domain (APP binding domain) that binds to an antiphagocytic protein (APP) (e.g., expressed on myeloid cells or on cells targeted for the death or inhibition of undesirable activity), (2) at least one binding domain (shielding domain) that conditionally inhibits the interaction between the anti-APP binding domain and its target, and (3) a proteolytically cleavable linker that, upon cleavage, removes the inhibition of APP binding by the shielding domain. The therapeutic conjugates encompassed by this disclosure may include one or more additional elements (e.g., one or more elements that promote and / or activate myeloid cells), and / or at least one binding domain (target cell binding domain) that binds to a target cell antigen. In some embodiments, a proteolytically cleavable linker is cleaved by a protease characteristic and / or specific to a particular target environment (e.g., a tumor microenvironment), thereby achieving activatable activity.This disclosure includes the finding that therapeutic conjugates according to this disclosure can selectively lead to the elimination of target cells by antibody-dependent cell-mediated cytotoxicity (ADCC) by directing myeloid cells in the tumor environment to target and / or phagocytose target cells, and / or by the activity of the therapeutic conjugates that directly kill target cells. In various embodiments, the target cells are cancer cells, and the therapeutic conjugates provided herein are useful in the treatment of cancer. In various embodiments, the therapeutic conjugates provided herein modulate the activity of target cells in a particular target environment (e.g., inhibiting undesirable activity, such as inhibiting inflammatory and / or autoimmune function of immune cell subtypes and / or antigen-specific immune cells).

[0005] The methods and compositions encompassed by this disclosure are associated with many advantages. For example, this disclosure recognizes that while blocking APP can direct the activity of myeloid cells (e.g., phagocytic activity) towards target cells of interest (such as cancer cells), such blocking can be harmful and / or toxic by directing myeloid cells in healthy tissue to target and destroy healthy cells. This challenge is particularly significant because a large amount of APP is present (e.g., ubiquitous) on both normal cells and target cells of interest (such as cancer cells). The various therapeutic conjugates encompassed by this disclosure include a shielding domain bound to a proteolytically cleavable linker, and the shielding domain is configured to maintain its inhibition of APP binding until the inhibition by the APP-binding domain is removed by cleavage of the linker. Therefore, the various therapeutic conjugates encompassed by this disclosure advantageously provide selective targeting of APP-expressing (e.g., myeloid) cells in a target environment of interest (e.g., tumor microenvironment) to direct myeloid cell activity (e.g., phagocytic activity) towards target cells (e.g., cancer cells). This selective targeting of myeloid cells in the target environment preserves healthy cells and improves pharmacokinetic properties (e.g., therapeutic index), safety, and / or efficacy against target cells of interest (e.g., cancer cells).

[0006] In some embodiments, the therapeutic conjugates of the Disclosure induce the elimination of target cells by antibody-dependent cell phagocytosis (ADCP). In some embodiments, the therapeutic conjugates of the Disclosure induce the elimination of target cells by antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the therapeutic conjugates of the Disclosure induce the elimination of target cells by direct death (also referred to herein as direct elimination). In some embodiments, the therapeutic conjugates encompassed by the Disclosure induce or promote the activity of myeloid cells selected from the group consisting of phagocytosis (e.g., antibody-dependent cell phagocytosis (ADCP)), direct cell-mediated cell death activity (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), those that promote lymphocyte-mediated cytotoxicity, etc.), and indirect cell death activity (e.g., those that promote complement-dependent cell-mediated cytotoxicity (CDC), those that reduce lymphocyte immune checkpoint inhibition, those that reduce myeloid cell immune checkpoint inhibition, etc.) (e.g., conditionally induce or promote myeloid cell activity to a target cell population). In some embodiments, the therapeutic conjugates encompassed by this disclosure induce or enhance specific myeloid cell activities, such as 1) phagocytosis but neither direct nor indirect cell-mediated cell death activity, 2) direct cell-mediated cell death activity but neither phagocytosis nor indirect cell death activity, and 3) indirect cell death activity but neither phagocytosis nor direct cell-mediated cell death activity (e.g., conditionally induce or enhance myeloid cell activity toward a target cell population). In some embodiments, the therapeutic conjugates encompassed by this disclosure involve regulating myeloid cell activity toward target cells (e.g., conditionally induced myeloid cell activity toward a target cell population) to inhibit undesirable activity (e.g., inhibiting the pro-inflammatory and / or autoimmune functions of immune cell subtypes and / or antigen-specific immune cells). In some embodiments, the therapeutic conjugates encompassed by this disclosure modulate the activity of myeloid cells relative to target cells (e.g., conditionally induced myeloid cell activity relative to a target cell population) to promote desired activity (such as promoting the anti-inflammatory and / or anti-autoimmune functions of immune cell subtypes and / or antigen-specific immune cells).

[0007] In at least certain aspects, the Disclosure provides a multispecific antigen-binding construct comprising (i) a first antigen-binding domain that binds to and inhibits an antiphagocytic protein (APP), and (ii) a second antigen-binding domain that binds to the first antigen-binding domain and inhibits or reduces the interaction between the first antigen-binding domain and APP, wherein the first and second antigen-binding domains are linked by a proteolytically cleavable linker.

[0008] In at least certain aspects, the Disclosure provides a multispecific antigen-binding construct comprising (i) a first antigen-binding domain that binds to and inhibits an antiphagocytic protein (APP), and (ii) a second antigen-binding domain that binds to the first antigen-binding domain, wherein the first and second antigen-binding domains are linked by a proteolytically cleavable linker, and when the linker is uncleaved, the second antigen-binding domain inhibits or reduces the binding of the first antigen-binding domain to APP, and when the linker is proteolytically cleaved, the second antigen-binding domain does not interfere with the binding of the first antigen-binding domain to APP.

[0009] In at least certain aspects, this disclosure is, (i) A first antigen-binding domain that binds to and inhibits antiphagocytic protein (APP), (ii) A second antigen-binding domain that binds to the first antigen-binding domain and inhibits or reduces the interaction between the first antigen-binding domain and APP, (iii) A linker containing a linker that can be cleaved in a proteolytic manner, (iv) A third antigen-binding domain that binds to the first target cell antigen, (v) Immunoglobulin Fc region, It provides a multispecific antigen-binding construct, and the linker, which includes a proteolytically cleavable linker, ligates a second antigen-binding domain to a first antigen-binding domain or an immunoglobulin Fc region.

[0010] In various embodiments, the first antigen-binding domain and the second antigen-binding domain are linked by a linker containing a proteolytically cleavable linker. In various embodiments, the immunoglobulin Fc region and the second antigen-binding domain are linked by a linker containing a proteolytically cleavable linker. In various embodiments, when the linker is uncleaved, the second antigen-binding domain inhibits or reduces the binding of the first antigen-binding domain to APP, and when the linker is proteolytically cleaved, the second antigen-binding domain does not interfere with the binding of the first antigen-binding domain to APP.

[0011] In various embodiments, APP includes, or is selected from, the group comprising, differentiation antigen group 47 (CD47), differentiation antigen group 24 (CD24), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 1 2 (PD-L2), β2-microglobulin (B2M), major histocompatibility complex class I (MHC-I), programmed cell death 1 (PD-1), signal regulatory protein α (SIRPα), sialic acid-binding immunoglobulin-like lectin 10 (SIGLEC10), leukocyte immunoglobulin-like receptor 1 (LILRB1), and leukocyte immunoglobulin-like receptor 2 (LILRB2). In various embodiments, the first antigen-binding domain either (1) inhibits the interaction between APP and its binding partner on myeloid cells, or (2) inhibits the interaction between APP and its binding partner on cells targeted for regulation (e.g., cell death (e.g., by phagocytosis)). In various embodiments, myeloid cells include macrophages, dendritic cells, monocytes, neutrophils, tumor-associated macrophages (TAMs), tumor-infiltrating macrophages (TIMs), or myeloid-derived immunosuppressive cells (MDSCs). In various embodiments, the first antigen-binding domain inhibits interactions including, or selected from, the interactions between CD47 and SIRPα, CD24 and SIGLEC10, PD-1 and PD-1 ligand (PD-L1 or PD-L2), LILRB1 ligand (β2M or MHC-I complex) and LILRB1, and LILRB2 ligand and LILRB2. Optionally, the first antigen-binding domain is SIRPα or its domain or fragment, SIGLEC10 or its domain or fragment, PD-1 or its domain or fragment, LILRB1 or its domain or fragment, LILRB2 or its domain or fragment, PD-L1 or its domain or fragment, PD-L2 or its domain or fragment, CD47 or its domain or protein, CD24 or its domain or protein, β2M or its domain or protein, or an MHC-I complex protein (HLA-A, HLA-B, or HLA-C).

[0012] In various embodiments, APP is CD47. In various embodiments, the first antigen-binding domain binds to CD47 and inhibits the interaction between CD47 and wild-type SIRPα, and optionally, the first antigen-binding domain binds to wild-type cell surface-expressed CD47 and inhibits the interaction between wild-type cell surface-expressed CD47 and wild-type cell surface-expressed SIRPα. In various embodiments, the first antigen-binding domain includes (i) the extracellular domain (ECD) of the cell surface-expressed protein, (ii) a binding fragment of the ECD of the cell surface-expressed protein, or (iii) a variant of the ECD or binding fragment of the cell surface-expressed protein that has been modified to improve binding to APP. In various embodiments, the cell surface-expressed protein is wild-type SIRPα, and the first antigen-binding domain includes (i) the ECD of wild-type SIRPα, (ii) a binding fragment of wild-type SIRPα, or (iii) a variant of the ECD or binding fragment of wild-type SIRPα that has been modified to improve binding to APP, and APP is CD47. In various embodiments, the binding fragment comprises the immunoglobulin variable (V) region (domain 1) of the cell surface-expressed protein ECD (optionally, the ECD of wild-type SIRPα). In various embodiments, the first antigen-binding domain comprises the domain of wild-type SIRPα that binds to CD47, or a variant thereof that includes one or more amino acid substitutions in the domain of wild-type SIRPα that improves binding to CD47.

[0013] In at least certain embodiments, the Disclosure provides a multispecific antigen-binding construct comprising: (a) a first antigen-binding domain comprising one or more amino acid substitutions in the domain of wild-type SIRPα that binds to an antiphagocytic protein (APP), or (ii) a variant thereof comprising one or more amino acid substitutions in the domain of wild-type SIRPα that improves binding to APP, wherein APP is CD47; and (b) a second antigen-binding domain which is an anti-SIRPα antibody or antigen-binding fragment that binds to the first antigen-binding domain and inhibits or reduces the interaction between the first antigen-binding domain and APP, wherein the first and second antigen-binding domains are linked by a linker comprising a proteolytically cleavable linker.

[0014] In various embodiments, the first antigen-binding domain is 100–120 amino acids long, optionally 106–118 amino acids long, and more optionally 112–118 amino acids long. In various embodiments, the domain of wild-type SIRPα is the extracellular domain of SIRPα. In various embodiments, the domain of wild-type SIRPα is the immunoglobulin variable region (IgV). In various embodiments, wild-type SIRPα is wild-type human SIRPα. In various embodiments, the first antigen-binding domain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103. In various embodiments, the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103. In various embodiments, the first antigen-binding domain is the sequence shown in SEQ ID NO: 103 or includes it.

[0015] In various embodiments, the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 104. In various embodiments, the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 104. In various embodiments, the first antigen-binding domain is the sequence shown in SEQ ID NO: 104, or comprises it.

[0016] In various embodiments, the first antigen-binding domain is a variant SIRPα comprising one or more amino acid substitutions in the IgV domain of wild-type SIRPα that improve binding to CD47. In various embodiments, the variant SIRPα has a dissociation constant (K) of less than 100 nanomolar (nM), less than 10 nM, less than 1 nM, less than 100 picomolar (pM), less than 10 pM, or less than 1 pM, or any combination between the aforementioned. D ) binds to wild-type human CD47. In various embodiments, variant SIRPα has a dissociation constant (K) of less than 100 nanomolar (nM) and optionally 1nM to 100nM, 1nM to 75nM, 1nM to 50nM, 1nM to 25nM, 1nM to 10nM, 10nM to 100nM, 10nM to 75nM, 10nM to 50nM, 10nM to 25nM, 25nM to 100nM, 25nM to 75nM, 25nM to 50nM, or 50nM to 100nM, 50nM to 75nM, or 75nM to 100nM. D ) binds to wild-type human CD47. In various embodiments, the variant SIRPα has a dissociation constant (K) less than 1 nM, optionally 100 pM to 1 nM, 100 pM to 750 pM, 100 pM to 500 pM, 100 pM to 250 pM, 250 pM to 1 nM, 250 pM to 750 pM, 250 pM to 500 pM, 500 pM to 1 nM, 500 pM to 750 pM, or 750 pM to 1 nM. DIt binds to wild-type human CD47 at a dissociation constant (K) of less than 100 picomolar concentrations (pM). In various embodiments, the variant SIRPα has a dissociation constant (K) of less than 100 picomolar concentrations (pM). D It binds to wild-type human CD47 at ) . In various embodiments, variant SIRPα has a dissociation constant (K) of 1 pM to 100 pM, optionally 1 pM to 75 pM, 1 pM to 50 pM, 1 pM to 25 pM, 1 pM to 10 pM, 10 pM to 100 pM, 10 pM to 75 pM, 10 pM to 50 pM, 10 pM to 25 pM, 25 pM to 100 pM, 25 pM to 75 pM, 25 pM to 50 pM, or 50 pM to 100 pM, 50 pM to 75 pM, or 75 pM to 100 pM. D It binds to wild-type human CD47.

[0017] In various embodiments, one or more amino acid substitutions are selected from the group consisting of L4F or L4I or L4V, V6F or V6I or V6L, V27F or V27I or V27L (A27F or A27I or A27L), I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L or H56R, S66G or S66T or S66A (or L66G or L66T or L66A), K68R, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, at least one amino acid substitution is E54Q (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

[0018] In various embodiments, one or more amino acid substitutions include K53R, E54Q, and S66T (L66T) (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, one or more amino acid substitutions include V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I; or V6I, V27I (or A27I), I31F, E47L, K53R, E54Q, H56P, S66T (or L66T); or L4V, V6I, V27I (or A27 I) I31F, E47V, K53R, E54Q, H56P, V63I, S66T (or L66T), K68R, V92I; or V6I, V27I (or A27I), I31T, E47V, K53R, E54Q, H56P, S66G (or L66G), K68R, V92I, F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105. In various embodiments, the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105. In various embodiments, the first antigen-binding domain is the amino acid sequence shown in SEQ ID NO: 105, or comprises it.

[0019] In various embodiments, the first antigen-binding domain is deglycosylated. In various embodiments, at least one of the one or more amino acid substitutions is N80A (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, the first antigen-binding domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In various embodiments, the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In various embodiments, the first antigen-binding domain is or includes the amino acid sequence shown in SEQ ID NO: 10.

[0020] In various embodiments, the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27. In various embodiments, the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27. In various embodiments, the first antigen-binding domain is the amino acid sequence shown in SEQ ID NO: 27, or comprises it.

[0021] In various embodiments, the first antigen-binding domain is an anti-CD47 antibody or an antigen-binding fragment that binds to CD47. In various embodiments, the first antigen-binding domain is a single-domain antibody, a single-chain variable fragment (scFv), sc(Fv)2, Fab, Fv, Fav, F(ab')2, Fab', dsFv, Fde, or sdFv. In various embodiments, the first antigen-binding domain is a single-domain antibody that is VHH. In various embodiments, VHH is a camel heavy-chain antibody, a humanized VHH domain, an affinity-mature VHH domain, or a human VHH domain. In various embodiments, the first antigen-binding domain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 208. In various embodiments, the first antigen-binding domain includes the sequence shown in Sequence ID No. 208. In various embodiments, the second antigen-binding domain has a dissociation constant for binding to the first antigen-binding domain that is greater than the dissociation constant of the first antigen-binding domain for APP. In various embodiments, the dissociation constant (Kd) of the second antigen-binding domain for the first antigen-binding domain is at least 2, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times greater than the dissociation constant of the first antigen-binding domain for APP. In various embodiments, once the cleavable linker of the second antigen-binding domain is cleaved, it does not interfere with or compete with the first antigen-binding domain for binding to APP. In various embodiments, the second antigen-binding domain has a dissociation constant for binding to the first antigen-binding domain of 1 nM or greater, optionally 100 nM to 1 μM, 10 nM to 1 μM, or 1 nM to 1 μM. In various embodiments, the second antigen-binding domain has a dissociation constant for binding to the first antigen-binding domain of 1 nM or greater. In various embodiments, the second antigen-binding domain has a dissociation constant for binding to the first antigen-binding domain of 10 nM or greater. In various embodiments, the second antigen-binding domain has a dissociation constant for binding to the first antigen-binding domain of 100 nM or greater.In various embodiments, the second antigen-binding domain has a dissociation constant for binding to the first antigen-binding domain of 1 μM or more.

[0022] In various embodiments, the second antigen-binding domain is an antibody or antigen-binding fragment. In various embodiments, the second antigen-binding domain is an anti-SIRPα antibody or antigen-binding fragment. In various embodiments, the antibody or antigen-binding fragment is a single-domain antibody, a single-chain variable fragment (scFv), sc(Fv)2, Fab, Fv, Fav, F(ab')2, Fab', dsFv, Fde, or sdFv. In various embodiments, the second antigen-binding domain is a single-domain antibody that is VHH. In various embodiments, VHH is a camel heavy-chain antibody, a humanized VHH domain, an affinity-mature VHH domain, or a human VHH domain.

[0023] In various embodiments, VHH includes a complementarity-determining region 1 (CDR1) containing an amino acid sequence selected from SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, and 45; a complementarity-determining region 2 (CDR2) containing an amino acid sequence selected from SEQ ID NOs: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and a complementarity-determining region 3 (CDR3) containing an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In various embodiments, the VHH domain includes CDR1, CDR2, and CDR3 as shown in SEQ ID NOs. 37, 46, and 61, SEQ ID NOs. 38, 46, and 61, SEQ ID NOs. 39, 47, and 62, SEQ ID NOs. 40, 48, and 63, SEQ ID NOs. 41, 49, and 64, SEQ ID NOs. 37, 50, and 61, SEQ ID NOs. 42, 51, and 65, SEQ ID NOs. 43, 52, and 66, SEQ ID NOs. 37, 53, and 67, SEQ ID NOs. 44, 54, and 68, SEQ ID NOs. 43, 55, and 63, SEQ ID NOs. 40, 56, and 69, SEQ ID NOs. 37, 57, and 70, SEQ ID NOs. 40, 55, and 63, SEQ ID NOs. 41, 58, and 71, SEQ ID NOs. 43, 59, and 72, SEQ ID NOs. 37, 60, and 73, or SEQ ID NOs. 45, 56, and 73, respectively. In various embodiments, the VHH domain includes an amino acid sequence shown in any one of SEQ ID NOs: 13-21 and 28-36, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 13-21 and 28-36, and binds to SIRPα. In various embodiments, the VHH domain includes an amino acid sequence shown in any one of SEQ ID NOs: 13-21 and 28-36.

[0024] In various embodiments, the VHH domain binds to the IgV domain of wild-type human SIRPα or a variant thereof. In various embodiments, the anti-SIRPα antibody or antigen-binding fragment is panreactive and binds to wild-type SIRPα and at least one variant SIRPα that includes one or more amino acid substitutions in the IgV domain of wild-type SIRPα that improves binding to CD47. In various embodiments, at least one variant SIRPα includes one or more amino acid substitutions in the IgV domain of wild-type SIRPα that improves binding to CD47. In various embodiments, one or more IgV domains or variants of wild-type human SIRPα are: (i) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103; (ii) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 104; (iii) amino acid sequences having at least 85%, 86%, 87%, 88% sequence identity with SEQ ID NO: 105 (iv) an amino acid sequence having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (iv) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (v

[0025] In various embodiments, the anti-SIRPα antibody or antigen-binding fragment binds to (1) the IgV domain of wild-type allele SIRPα, optionally to the IgV domain of wild-type allele 1 and / or wild-type allele 2 SIRPα, and (2) to at least one IgV domain of variant SIRPα containing one or more amino acid substitutions in the IgV domain of wild-type SIRPα that improves binding to CD47. In various embodiments, the anti-SIRPα antibody or antigen-binding fragment binds to wild-type human SIRPα, optionally to the IgV domain of wild-type human SIRPα, and wild-type human SIRPα has an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103. (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 104, or (ii) a wild-type human SIRPα having the amino acid sequence shown in sequence number 104.

[0026] In various embodiments, the anti-SIRPα antibody or antigen-binding fragment binds to variant SIRPα, and optionally binds to the IgV domain of variant SIRPα, and variant SIRPα is L4F or L4I or L4V, V6F or V6I or V6L, V27F or V27I or V27L (A27F or A27I or A27L), I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54 The wild-type SIRPα includes one or more amino acid substitutions selected from the group consisting of Q or E54H, H56P or H56L or H56R, S66G or S66T or S66A (or L66G or L66T or L66A), K68R, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, the one or more amino acid substitutions include K53R, E54Q, and S66T (L66T) (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

[0027] In various embodiments, one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I; or V6I, V27I (or A27I), I31F, E47L, K53R, E54Q, H56P, S66T (or L66T); or L4V, V6I, V27I (or A27 I) I31F, E47V, K53R, E54Q, H56P, V63I, S66T (or L66T), K68R, V92I; or V6I, V27I (or A27I), I31T, E47V, K53R, E54Q, H56P, S66G (or L66G), K68R, V92I, F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, variant SIRPα is named FB3, FD6, FA4, or CV1.

[0028] In various embodiments, an anti-SIRPα antibody or antigen-binding fragment binds to variant SIRPα, and optionally to the IgV domain of variant SIRPα, and these bind to (i) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105, or (ii) at least (iii) an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or (iii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27.

[0029] In various embodiments, the anti-SIRPα antibody or antigen-binding fragment has a dissociation constant (K) of wild-type human SIRPα or its variant against wild-type human CD47. D CD47 binds to wild-type human SIRPα or its variants with a KD at least 2, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times greater than ), and optionally, CD47 is cell surface-expressed CD47.

[0030] In various embodiments, the linker is a substrate of a protease, optionally, the protease is an extracellular protease, and / or the linker is a substrate of renin, pepsin C, napsin A, matrix metalloproteinase (MMP), matryptase, urokinase-type plasminogen activator (uPA), disintegrin and metalloproteinase (ADAM), disintegrin and metalloproteinase having a thrombospondin motif (ADAMTS), regmine, urokinase, or hepsin.

[0031] In various embodiments, the proteolytically cleavable linker is a polypeptide that functions as a substrate for the protease. In various embodiments, the protease is produced by a tumor or by cells present in the tumor microenvironment. In various embodiments, the protease is selected from matryptase, matrix metalloproteinase (MMP), granzyme B, and combinations thereof. In various embodiments, the protease is matryptase. In various embodiments, the proteolytically cleavable linker is VHMPLGFLGPRQARVVN (SEQ ID NO: 22). In various embodiments, the linker containing the proteolytically cleavable linker includes an N-terminal and / or C-terminal GS linker sequence. In various embodiments, the GS linker sequence is the sequence (GGGGS)n (where n is 1 to 5) (SEQ ID NO: 259), and optionally, the GS linker sequence is GGGGSGGGGS (SEQ ID NO: 9) or GGGGS (SEQ ID NO: 11).

[0032] In various embodiments, the construct further comprises a third antigen-binding domain that binds to a first target cell antigen, and optionally, the first target cell antigen is expressed on cells targeted for regulation (e.g., death (e.g., by phagocytosis)). In various embodiments, the first target cell antigen is expressed on cells targeted for myeloid cell activity. In various embodiments, the third antigen-binding domain is an antibody or antigen-binding fragment. In various embodiments, the antibody or antigen-binding fragment is a single-strand variable fragment (scFv), sc(Fv)2, Fab, Fv, Fav, F(ab')2, Fab', dsFv, Fde, sdFv, or a single-domain antibody (sdAb).

[0033] In various embodiments, the first target cell antigen is a microbial antigen, a peptide-major histocompatibility complex (pMHC), or a tumor-associated antigen (TAA), and optionally, the TAA is selected from the group of TAAs listed in Table 2 or derived from the targets listed in Table 2.

[0034] In various embodiments, the third antigen-binding domain is Fab. In various embodiments, the third antigen-binding domain is a single-chain antibody fragment. In various embodiments, the third antigen-binding domain is VHH. In various embodiments, VHH is a camel heavy-chain antibody, a humanized VHH domain, an affinity-mature VHH domain, or a human VHH domain. In various embodiments, the third antigen-binding domain is a single-chain variable fragment (scFv). In various embodiments, the third antigen-binding domain contains two different single-chain antibody fragments. In various embodiments, the third antigen-binding domain is a biparatopic. In various embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain are antibodies. In various embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain may be the extracellular domain (ECD) of a cell surface-expressed protein, a fragment of the ECD of a cell surface-expressed protein, a variant version of the ECD of a cell surface-expressed protein manipulated to improve binding to a target, an antibody-binding domain, a functional fragment of an antibody, its variable domain, a VH domain, a VL domain, a VNAR domain, a VHH domain, a single-strand variable fragment (scFv), sc(Fv)2, Fab, Fv, Fav, F(ab')2, Fab', dsFv, Fde, sdFv, a single-domain antibody (sdAb), a nanobody, a bispecific antibody, a diabody, an intrabody, or a domain antibody. This includes bodies, antibody mimes, zybodies, polypeptide-Fc fusions, camel antibodies, camelized antibodies, shielded antibodies (affybodies), anti-idiotype (anti-Id) antibodies, single-stranded diabodies, tandem diabodies, VHH, antikalin, minibodies, BiTE, ankyrin repeat proteins, DARPIN, avimer, DART, TCR-like antibodies, adonectin, affilin, transbodies, affibodies, TrimerX, microproteins, finomers, centyrin, KALBITOR, CAR, engineered TCRs, or functional fragments or combinations thereof.In various embodiments, the antibody comprises an immunoglobulin constant domain selected from the group comprising or consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. In various embodiments, the construct and / or antibody comprises a constant domain derived from human immunoglobulin.

[0035] In various embodiments, the multispecific antigen-binding construct further comprises a fourth antigen-binding domain that binds to a second target cell antigen, and optionally, the second target cell antigen is expressed on a cell targeted for regulation (e.g., death (e.g., by phagocytosis)). In various embodiments, the fourth antigen-binding domain is an antibody or antigen-binding fragment. In various embodiments, the antibody or antigen-binding fragment is a single-strand variable fragment (scFv), sc(Fv)2, Fab, Fv, Fav, F(ab')2, Fab', dsFv, Fde, sdFv, or a single-domain antibody (sdAb). In various embodiments, the fourth antigen-binding domain is Fab. In various embodiments, the second target cell antigen is a microbial antigen, a peptide-major histocompatibility complex (pMHC), or a tumor-associated antigen (TAA). In various embodiments, the second target cell antigen is a TAA, which is selected from the group of TAAs listed in Table 2 or derived from the targets listed in Table 2.

[0036] In various embodiments, the multispecific antigen-binding construct further comprises an immunoglobulin Fc region. In various embodiments, the immunoglobulin Fc region is a homodimeric Fc region. In various embodiments, the third antigen-binding domain is bivalent. In various embodiments, the immunoglobulin Fc region is a wild-type human IgG1 Fc region. In various embodiments, the immunoglobulin Fc region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 98. In various embodiments, the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO: 98. In various embodiments, the Fc region is a variant Fc region containing one or more amino acid mutations or substitutions that increase the antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) activity of the multispecific antigen-binding construct. In various embodiments, the variant Fc region contains one or more amino acid mutations compared to the wild-type human IgG1 Fc region. In various embodiments, the variant Fc region is 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 251, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 272, 279, 280, 281, 282, 283, 284, 292, 293, 295, 296, 29 The mutant includes one or more mutations at positions selected from the group including or consisting of 7, 298, 299, 300, 304, 305, 309, 313, 316, 318, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 339, 341, 343, 370, 373, 378, 392, 396, 416, 419, 421, 440, and 443, and optionally, one or more of the mutations are substitutions, and optionally, the substitutions are 220S, 229S, 232G, 233P, 234A, 234D, 234E, 234F, 234G, 234H, 234L, 234N, 234Q, 234T, 234V, 234Y, 235A, 235D, 235E, 235F, 235 G, 235H, 235N, 235P, 235Q, 235R, 235S, 235T, 235W, 235Y, 236A, 236E, 236I, 236N, 236P, 236R, 237A, 237K, 237L, 237N, 237P, 23 8K, 238S, 239D, 239E, 239F, 239H, 239N, 239Q, 239R, 239T, 239Y, 240M, 240T, 241A, 241E, 241L, 241W, 241Y, 243L, 243Q, 243R, 2 43W, 243Y, 244H, 245A, 247G, 247I, 247L, 247V, 24IR, 252Y, 254T, 255L, 256E, 256M, 25IF, 262E, 262T, 263M, 263T, 264A, 264E,264F, 264L, 264M, 264R, 264T, 264Y, 265A, 265F, 265G, 265H, 265N, 265Q, 265T, 265V, 265Y, 266M, 266T, 267E267L, 267Q, 267R, 268E, 268Q, 269F, 269G, 269H, 269R, 269Y, 270E, 270H, 280A, 284M, 292L, 292P, 296D, 296E, 296L, 296N , 296Q, 296S, 296T, 297A, 297D, 297E, 297S, 298A, 298F, 298H, 299A, 299E, 299F, 299H, 299I, 299S, 299V, 300L, 305 I, 309L, 316D, 318A, 324T, 325A, 325E, 325H, 325L, 325Q, 325T, 325V, 326W, 327G, 327L, 327N, 327R, 327W, 328A, 32 8D, 328E, 328F, 328H, 328M, 328N, 328Q, 328R, 328S, 328T, 329F, 329H, 329K, 329Q, 330C, 330F, 330G, 330H, 330I, 3 30K, 330L, 330N, 330P, 330R, 330S, 330T, 330V, 330Y, 331A, 331D, 331E, 331F, 331G, 331H, 331K, 331L, 331M, 331N, The amino acid variants are selected from the group including or consisting of 331Q, 331R, 331S, 331T, 331V, 331W, 331Y, 332A, 332D, 332E, 332F, 332H, 332N, 332Q, 332S, 332T, 332W, 332Y, 333A, 333S, 334A, 339Q, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y, and 443W. In various embodiments, one or more amino acid mutations are the amino acid substitutions G236A, S239D, and I332E. In various embodiments, the immunoglobulin Fc region contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 97. In various embodiments, the immunoglobulin Fc region includes the amino acid sequence shown in SEQ ID NO: 97.

[0037] In various embodiments, the third antigen-binding region is Fab, and the multispecific antigen-binding construct comprises a first polypeptide chain comprising a heavy chain variable region (VH) and heavy chain constant region 1 (CH1) of Fab, an immunoglobulin Fc region, a first antigen-binding domain, a linker comprising a proteolytically cleavable linker, and a second antigen-binding domain, and a second polypeptide comprising a light chain variable region (VL) and light chain constant region (CL) of Fab. In various embodiments, the multispecific polypeptide construct comprises two identical first polypeptides and two identical second polypeptides, the two first polypeptides covalently linked by disulfide bonds, and each of the second polypeptides covalently linked to one of the first polypeptides by disulfide bonds. In various embodiments,

[0038] In various embodiments, the third antigen-binding region is Fab, and the multispecific antigen-binding construct comprises a first polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain variable region (VH) and a heavy chain constant region (CH1) of Fab, an immunoglobulin Fc region comprising the amino acid sequence shown in SEQ ID NO: 97, a first antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 10, a linker comprising a proteolytic cleavage linker shown in SEQ ID NO: 12, and a second antigen-binding domain comprising a sequence having at least 95% sequence identity with any one of the sequences shown in SEQ ID NOs: 13-21 and 28-36, and a second polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of Fab. In various embodiments, the third antigen-binding region is Fab, and the multispecific antigen-binding construct comprises a first polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain variable region (VH) and a heavy chain constant region (CH1) of Fab, an immunoglobulin Fc region comprising the amino acid sequence shown in SEQ ID NO: 97, a first antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 27, a linker comprising a proteolytic cleavage linker shown in SEQ ID NO: 12, and a second antigen-binding domain comprising a sequence having at least 95% sequence identity with any one of the sequences shown in SEQ ID NOs: 13-21 and 28-36, and a second polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of Fab.

[0039] In various embodiments, the second antigen-binding domain includes the sequence shown in any one of SEQ ID NOs: 13-21 and 28-36. In various embodiments, the multispecific antigen-binding construct includes a peptide linker between the immunoglobulin Fc region and the first antigen-binding domain. In various embodiments, the peptide linker is a GS linker. In various embodiments, the GS linker is (GGGGS)n (where n is 1-5 in the sequence) (SEQ ID NO: 259). In various embodiments, the GS linker is GGGGS (SEQ ID NO: 11), GGGGSGGGGS (SEQ ID NO: 9), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23), or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 24).

[0040] In various embodiments, the first target cell antigen is EGFR. In various embodiments, the third antigen-binding domain is a Fab derived from an antibody selected from the group consisting of necitumumab (11F8), cetuximab, nimotuzumab, and P2X. In various embodiments, Fab includes (a) a heavy chain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7 and a light chain having at least 95% sequence identity with SEQ ID NO: 2; (b) a heavy chain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 205 and a light chain having at least 95% sequence identity with SEQ ID NO: 2; (c) a heavy chain containing an amino acid sequence having at least 95% sequence identity with amino acids 1-217 of SEQ ID NO: 93 and a light chain having at least 95% sequence identity with SEQ ID NO: 94; (d) a heavy chain containing a sequence having at least 95% sequence identity with amino acids 1-221 of SEQ ID NO: 211 and a light chain having at least 95% sequence identity with SEQ ID NO: 96; or (e) a heavy chain containing a sequence having at least 95% sequence identity with amino acids 1-217 of SEQ ID NO: 212 and a light chain having at least 95% sequence identity with SEQ ID NO: 213. In various embodiments, Fab includes (a) a heavy chain containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2, (b) a heavy chain containing the amino acid sequence shown in SEQ ID NO: 205 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2, (c) a heavy chain containing amino acids 1 to 217 of SEQ ID NO: 93 and a light chain containing the amino acid sequence shown in SEQ ID NO: 94, (d) a heavy chain containing amino acids 1 to 221 of SEQ ID NO: 211 and a light chain containing the amino acid sequence shown in SEQ ID NO: 96, or (e) a heavy chain containing amino acids 1 to 217 of SEQ ID NO: 212 and a light chain containing the amino acid sequence shown in SEQ ID NO: 213.

[0041] In various embodiments, Fab is a necitumumab Fab comprising a heavy chain containing a sequence having at least 95% sequence identity with SEQ ID NO: 7 and a light chain containing at least 95% sequence identity with SEQ ID NO: 2. In various embodiments, Fab is a necitumumab Fab comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2. In various embodiments, Fab is a necitumumab Fab comprising a heavy chain containing a sequence having at least 95% sequence identity with SEQ ID NO: 205 and a light chain containing at least 95% sequence identity with SEQ ID NO: 2. In various embodiments, Fab is a necitumumab Fab comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 205 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2.

[0042] In various embodiments, the multispecific polypeptide construct includes a first polypeptide chain containing a sequence having at least 95% sequence identity with the sequence shown in any one of SEQ ID NOs: 110-120, and a second polypeptide chain containing a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 2. In various embodiments, the multispecific polypeptide construct includes a first polypeptide chain containing a sequence showing in any one of SEQ ID NOs: 110-120, and a second polypeptide chain containing a sequence showing in SEQ ID NO: 2. In various embodiments, the multispecific polypeptide construct includes a first polypeptide chain containing a sequence having at least 95% sequence identity with the sequence shown in any one of SEQ ID NOs: 125-137, and a second polypeptide chain containing a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 2. In various embodiments, the multispecific polypeptide construct includes a first polypeptide chain containing a sequence showing in any one of SEQ ID NOs: 125-137, and a second polypeptide chain containing a sequence showing in SEQ ID NO: 2.

[0043] In various embodiments, the third antigen-binding region is a single-chain antibody fragment, and the multispecific antigen-binding construct comprises a polypeptide comprising the third antigen-binding region, an Fc region, a first antigen-binding domain, a linker containing a proteolytically cleavable linker, and a second antigen-binding domain. In various embodiments, the third antigen-binding region is a single-chain antibody fragment, and the multispecific antigen-binding construct comprises the third antigen-binding region, an Fc region, a first antigen-binding domain, a linker containing a proteolytically cleavable linker, and a second antigen-binding domain, in order from the N-terminus to the C-terminus.

[0044] In various embodiments, the immunoglobulin Fc region is a variant Fc region comprising a modified hinge domain including the substitution of amino acids EPKSC to EPKSS. In various embodiments, the immunoglobulin Fc region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102. In various embodiments, the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO: 102.

[0045] In various embodiments, the first target cell antigen is EGFR. In various embodiments, the third antigen-binding domain is an scFv derived from a selection of necitumumab (11F8), cetuximab, nimotuzumab, and P2X. In various embodiments, the third antigen-binding domain is an scFv derived from necitumumab (11F8). In various embodiments, the scFv includes a variable heavy (VH) chain containing an amino acid sequence having at least 95% sequence identity with the VH chain sequence present in SEQ ID NO: 207, and a variable light (VL) chain containing an amino acid sequence having at least 95% sequence identity with the VL chain sequence present in SEQ ID NO: 207. In various embodiments, the scFv includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 207. In various embodiments, the scFv includes the amino acid sequence shown in SEQ ID NO: 207.

[0046] In various embodiments, the Fc region is a heterodimeric Fc region. In various embodiments, the first antigen-binding domain or the third antigen-binding domain is divalent, and the first antigen-binding domain and the other of the third antigen-binding domain are monovalent. In various embodiments, the first antigen-binding domain is divalent, and the third antigen-binding domain is monovalent. In various embodiments, the first antigen-binding domain is monovalent, and the third antigen-binding domain is divalent.

[0047] In various embodiments, the multispecific antigen-binding construct comprises a first antigen-binding domain that binds to an antiphagocytic protein (APP), a second antigen-binding domain that binds to the first antigen-binding domain, a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, and a third antigen-binding domain which is a target cell antigen-binding domain that binds to a target cell antigen expressed on cells targeted for myeloid cell activity, wherein the second antigen-binding domain is linked to either the first or second Fc polypeptide by a proteolytically cleavable linker. In various embodiments, the multispecific antigen-binding construct comprises (1) a first heavy chain comprising a first polypeptide chain and a first antigen-binding domain of heterodimeric Fc, and (2) a second heavy chain comprising a second polypeptide chain of heterodimeric Fc, a linker comprising a proteolytically cleavable linker, and a second antigen-binding domain, wherein at least one or both of the first and second heavy chains comprise the third antigen-binding domain or its chain. In various embodiments, the third antigen-binding domain is Fab, and each of the first and second heavy chains comprises a variable heavy (VH) chain and CH1 of Fab. In various embodiments, the multispecific antigen-binding construct further comprises a light chain containing a light chain (VL-CL) of Fab of the third antigen-binding domain.

[0048] In various embodiments, when the third antigen-binding domain is Fab, the multispecific antigen-binding construct comprises: a first polypeptide containing the heavy chain variable region (VH) and heavy chain constant region 1 (CH1) of Fab, a first polypeptide of the heterodimeric immunoglobulin Fc region, and the first antigen-binding domain in N-terminus to C-terminus; a second polypeptide containing the VH and CH1 of Fab, the heterodimeric Fc region, a linker containing a proteolytically cleavable linker, and the second antigen-binding domain in N-terminus to C-terminus; and a third polypeptide containing the light chain variable region (VL) and light chain constant region (CL) of Fab.

[0049] In various embodiments, at least one Fc polypeptide of the heterodimer Fc region, optionally each Fc polypeptide, comprises at least one amino acid substitution to promote heterodimerization compared to the polypeptide of the homodimer Fc region, and optionally compared to the IgG1 Fc region. In various embodiments, one or more amino acid substitutions are either knob-into-hole modifications or charge mutations to increase the electrostatic complementarity of the polypeptide. In various embodiments, the first Fc polypeptide of the heterodimer Fc region includes an amino acid substitution selected from Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof, and the second Fc polypeptide of the heterodimer Fc region includes an amino acid substitution T366W. Optionally, the first and second Fc polypeptides further include an amino acid substitution from a non-cysteine ​​residue to a cysteine ​​residue, where the amino acid substitution of the first polypeptide is at either Ser354 or Y349, and the amino acid substitution of the second Fc polypeptide is at either Ser354 or the other Y349. In various embodiments, the first Fc polypeptide includes the amino acid substitutions Y349C, T366S, L368A, and Y407V, and the second Fc polypeptide includes the amino acid substitutions S354C and T366W. In various embodiments, the first Fc polypeptide comprises the amino acid sequence shown in SEQ ID NO: 100, and the second Fc polypeptide comprises the amino acid sequence shown in SEQ ID NO: 101. In various embodiments, the first Fc polypeptide comprises the amino acid sequence shown in SEQ ID NO: 106, and the second Fc polypeptide comprises the amino acid sequence shown in SEQ ID NO: 107.

[0050] In various embodiments, the multispecific antigen-binding construct includes a first polypeptide comprising, from N-terminus to C-terminus, the heavy chain variable region (VH) and heavy chain constant region (CH1) of Fab, a first immunoglobulin Fc region containing the amino acid sequence shown in SEQ ID NO: 106, and a first antigen-binding domain containing the amino acid sequence shown in SEQ ID NO: 27; a second polypeptide comprising, from N-terminus to C-terminus, the VH and CH1 of Fab, a second immunoglobulin Fc region containing the amino acid sequence shown in SEQ ID NO: 107, a linker containing a proteolytic cleavage linker shown in SEQ ID NO: 12, and a second antigen-binding domain containing a sequence having at least 95% sequence identity with the sequence shown in any one of SEQ ID NOs. 13-21 and 28-36; and a third polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of Fab. In various embodiments, the second antigen-binding domain contains the sequence shown in any one of SEQ ID NOs. 13-21 and 28-36.

[0051] In various embodiments, the first target cell antigen is EGFR. In various embodiments, the third antigen-binding domain is a Fab derived from an antibody selected from the group consisting of necitumumab (11F8), cetuximab, nimotuzumab, and P2X. In various embodiments, Fab includes (a) a heavy chain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7 and a light chain having at least 95% sequence identity with SEQ ID NO: 2; (b) a heavy chain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 205 and a light chain having at least 95% sequence identity with SEQ ID NO: 2; (c) a heavy chain containing an amino acid sequence having at least 95% sequence identity with amino acids 1-217 of SEQ ID NO: 93 and a light chain having at least 95% sequence identity with SEQ ID NO: 94; (d) a heavy chain containing a sequence having at least 95% sequence identity with amino acids 1-221 of SEQ ID NO: 211 and a light chain having at least 95% sequence identity with SEQ ID NO: 96; or (e) a heavy chain containing a sequence having at least 95% sequence identity with amino acids 1-217 of SEQ ID NO: 212 and a light chain having at least 95% sequence identity with SEQ ID NO: 213. In various embodiments, Fab includes (a) a heavy chain containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2, (b) a heavy chain containing the amino acid sequence shown in SEQ ID NO: 205 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2, (c) a heavy chain containing amino acids 1 to 217 of SEQ ID NO: 93 and a light chain containing the amino acid sequence shown in SEQ ID NO: 94, (d) a heavy chain containing amino acids 1 to 221 of SEQ ID NO: 211 and a light chain containing the amino acid sequence shown in SEQ ID NO: 96, or (e) a heavy chain containing amino acids 1 to 217 of SEQ ID NO: 212 and a light chain containing the amino acid sequence shown in SEQ ID NO: 213.

[0052] In various embodiments, Fab is a necitumumab Fab comprising a heavy chain containing a sequence having at least 95% sequence identity with SEQ ID NO: 7 and a light chain containing at least 95% sequence identity with SEQ ID NO: 2. In various embodiments, Fab is a necitumumab Fab comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2. In various embodiments, Fab is a necitumumab Fab comprising a heavy chain containing a sequence having at least 95% sequence identity with SEQ ID NO: 205 and a light chain containing at least 95% sequence identity with SEQ ID NO: 2. In various embodiments, Fab is a necitumumab Fab comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 205 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2.

[0053] In at least certain embodiments, the Disclosure provides nucleic acids encoding multispecific antigen-binding constructs according to the Disclosure. In at least certain embodiments, the Disclosure provides expression vectors comprising the nucleic acids of the Disclosure. In at least certain embodiments, the Disclosure provides cells comprising the expression vectors of the Disclosure. In at least certain embodiments, the Disclosure provides a method for producing multispecific antigen-binding constructs, the method comprising culturing cells or populations of such cells under conditions that promote the expression of multispecific antigen-binding constructs from expression vectors by the cells. In various embodiments, the method for producing multispecific antigen-binding constructs provided herein further includes isolating the multispecific antigen-binding constructs from cells or populations of cells, or from a medium in which cells or populations of cells were cultured. In at least certain embodiments, the Disclosure provides a pharmaceutical composition comprising the multispecific antigen-binding constructs of the Disclosure and a pharmaceutically acceptable carrier or excipient.

[0054] In at least certain embodiments, the Disclosure provides a method for increasing the regulation of target cells by myeloid cells (e.g., death (e.g., by phagocytosis)), the method comprising contacting a population of target cells with a sufficient amount of the multispecific antigen-binding construct of the Disclosure to regulate or increase the regulation of target cells by myeloid cells (e.g., death (e.g., by phagocytosis)) in the presence of myeloid cells. In various embodiments, the myeloid cells are macrophages, dendritic cells, neutrophils, tumor-associated macrophages (TAMs), or tumor-infiltrating macrophages (TIMs). In various embodiments, the target cells are infected with a microorganism or express a microbial antigen. In various embodiments, the microbial antigen is a viral antigen. In various embodiments, the cells are cancer cells, or the cells express tumor-associated antigens (TAAs). In various embodiments, the TAAs are selected from the group of TAAs listed in Table 2, or derived from the targets listed in Table 2.

[0055] In at least certain embodiments, the Disclosure provides a method for treating a subject having a microbial infection, the method comprising administering an effective amount of the multispecific antigen-binding construct of the Disclosure to the subject, thereby treating the microbial infection in the subject. In at least certain embodiments, the Disclosure provides a method for treating or delaying the progression of cancer in a subject, the method comprising administering an effective amount of the multispecific antigen-binding construct of the Disclosure to the subject, thereby treating and / or delaying the progression of cancer in the subject. In various embodiments, cancer is adenocarcinoma, cholangiocarcinoma (biliary tract cancer), bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, colon cancer, endometrial cancer, esophageal cancer, glioma, head and neck cancer, head and neck squamous cell carcinoma, leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer, kidney cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, or urothelial carcinoma. In various embodiments, the method further comprises administering an additional therapeutic agent to treat the cancer. In various embodiments, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint inhibitor.

[0056] In at least certain aspects, the Disclosure provides a method for treating or delaying the progression of an autoimmune or inflammatory disease in a subject, the method comprising administering an effective amount of the multispecific antigen-binding construct of the Disclosure to the subject, thereby treating and / or delaying the autoimmune or inflammatory disease in the subject.In various embodiments, autoimmune or inflammatory diseases include atherosclerosis, obesity, inflammatory bowel disease (IBD), Lyme disease, Hashimoto's thyroiditis, autoimmune uveitis, autoimmune heart valve disease, rheumatoid arthritis, allergic encephalitis, atopic dermatitis, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, Sjögren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylitis, type 1 diabetes mellitus, alopecia areata, vasculitis, and temporal arteritis, graft-versus-host disease (GVHD), asthma, COPD, eosinophilia, conjunctivitis, glomerulonephritis, autoimmune nephritis, paraneoplastic autoimmune diseases, chondritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarthritis of juvenile arthritis Rheumatism, polyarticular juvenile rheumatoid arthritis, systemic juvenile juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, myotendonitis, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, fibrous disease, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-joint rheumatoid arthritis, systemic rheumatoid arthritis, enteroarthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodosa Nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, psoriasis, psoriasis vulgaris, guttate psoriasis, reverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, Crohn's disease, ulcerative colitis, celiac disease, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplant rejection, kidney injury, hepatitis C-induced vasculitis, viral infection, bacterial infection, or spontaneous remission of pregnancy.

[0057] In at least certain embodiments, the Disclosure provides a method for treating or delaying the progression of a cardiovascular disease in a subject, the method comprising administering an effective amount of the multispecific antigen-binding construct of the Disclosure to the subject, thereby treating and / or delaying the progression of a cardiovascular disease in the subject. In various embodiments, the cardiovascular disease is atherosclerosis, stroke, coronary artery disease, cerebrovascular disease, congenital heart disease, peripheral vascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, cor pulmonale, arrhythmia, endocarditis, myocarditis, eosinophilic myocarditis, valvular heart disease, congenital heart disease, or rheumatic heart disease.

[0058] In at least certain embodiments, the Disclosure provides a method for treating or delaying the progression of a neurological disorder in a subject, the method comprising administering an effective amount of the multispecific antigen-binding construct of the Disclosure to the subject, thereby treating and / or delaying the progression of the neurological disorder in the subject. In various embodiments, the neurological disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, ophthalmic disorders, glaucoma, myotonic dystrophy, Guillain-Barré syndrome (GBS), myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, traumatic brain injury (TBI), epilepsy, or Huntington's disease (HD).

[0059] In various methods of this disclosure, the subject is a mammal, for example, a mammal is a human, a non-human primate, a farm animal, a livestock, or a laboratory animal. In various methods of this disclosure, the multispecific antigen-binding construct is administered orally, rectally, intravenously, intratumorally, or subcutaneously.

[0060] In at least certain embodiments, the Disclosure provides a SIRPα-binding molecule comprising at least one heavy-chain-only variable domain (SIRPα VHH domain), which includes: Complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, and 45; Complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from SEQ ID NOs: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and Complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In various embodiments, at least one SIRPα The VHH domains include CDR1, CDR2, and CDR3 as shown in SEQ ID NOs. 37, 46, and 61, SEQ ID NOs. 38, 46, and 61, SEQ ID NOs. 39, 47, and 62, SEQ ID NOs. 40, 48, and 63, SEQ ID NOs. 41, 49, and 64, SEQ ID NOs. 37, 50, and 61, SEQ ID NOs. 42, 51, and 65, SEQ ID NOs. 43, 52, and 66, SEQ ID NOs. 37, 53, and 67, SEQ ID NOs. 44, 54, and 68, SEQ ID NOs. 43, 55, and 63, SEQ ID NOs. 40, 56, and 69, SEQ ID NOs. 37, 57, and 70, SEQ ID NOs. 40, 55, and 63, SEQ ID NOs. 41, 58, and 71, SEQ ID NOs. 43, 59, and 72, SEQ ID NOs. 37, 60, and 73, or SEQ ID NOs. 45, 56, and 73, respectively. In various embodiments, SIRPα is human SIRPα. In various embodiments, at least one SIRPα VHH domain contains an amino acid sequence shown in any one of SEQ ID NOs. 13-21 and 28-36, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 13-21 and 28-36, and binds to SIRPα.In various embodiments, at least one SIRPα VHH domain contains the amino acid sequence shown in any one of SEQ ID NOs: 13-21 and 28-36.

[0061] In various embodiments, binding of the SIRPα VHH domain to SIRPα inhibits or reduces the binding of SIRPα to differentiation antigen group 47 (CD47). In various embodiments, the binding affinity of the SIRPα VHH domain to SIRPα is higher than the binding affinity of SIRPα to CD47. In various embodiments, the VHH domain binds to the IgV domain or variant of one or more wild-type human SIRPα. In various embodiments, one or more IgV domains or variants of wild-type human SIRPα have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (i) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (iii) an amino acid sequence having at least 85%, 86%, 87%, 8 (iv) an amino acid sequence having sequence identity of %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, selected from (iv) an amino acid sequence having sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with sequence number 10, and (v) an amino acid sequence having sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with sequence number 2.

[0062] In various embodiments, VHH binds to at least one variant SIRPα that is panreactive and contains one or more amino acid substitutions in the IgV domain of wild-type SIRPα that improves binding to wild-type SIRPα and CD47. In various embodiments, VHH binds to (1) the IgV domain of wild-type allele SIRPα, optionally the IgV domain of wild-type allele 1 and / or wild-type allele 2 SIRPα, and (2) at least one IgV domain of variant SIRPα that (a) contains one or more amino acid substitutions in the IgV domain of wild-type SIRPα that improves binding to CD47, and / or (b) is a deglycosylated variant.

[0063] In various embodiments, the IgV domain of wild-type human SIRPα, optionally, includes (i) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103, or (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 104. In various embodiments, the IgV domain of variant SIRPα may be L4F or L4I or L4V, V6F or V6I or V6L, V27F or V27I or V27L (A27F or A27I or A27L), I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L, and Alternatively, it includes one or more amino acid substitutions in wild-type SIRPα, selected from the group consisting of H56R, S66G or S66T or S66A (or L66G or L66T or L66A), K68R, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, one or more amino acid substitutions include K53R, E54Q, and S66T (L66T) (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).In various embodiments, one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I; or V6I, V27I (or A27I), I31F, E47L, K53R, E54Q, H56P, S66T (or L66T); or L4V, V6I, V27I (or A27 I) I31F, E47V, K53R, E54Q, H56P, V63I, S66T (or L66T), K68R, V92I; or V6I, V27I (or A27I), I31T, E47V, K53R, E54Q, H56P, S66G (or L66G), K68R, V92I, F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In various embodiments, one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

[0064] In various embodiments, variant SIRPα is FB3, FD6, FA4, or CV1. In various embodiments, variant SIRPα is optionally the IgV domain of variant SIRPα having (i) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105, or (ii) at least 85%, 86%, 87%, 88% sequence identity with SEQ ID NO: 10 (iii) an amino acid sequence having sequence identity of %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or (iii) an amino acid sequence having sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with sequence number 27.

[0065] In at least certain embodiments, the Disclosure provides a binding molecule comprising a SIRPα-binding molecule according to the Disclosure and a second binding domain that binds to a second antigen. In various embodiments, the second antigen is a tumor antigen. In various embodiments, the tumor antigen is a TAA selected from the group of tumor-associated antigens (TAAs) listed in Table 2, or a TAA derived from a target listed in Table 2. In various embodiments, the tumor antigens are CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), EGFR, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3). In various embodiments, the binding domain that binds to the second antigen is an antibody or an antigen-binding fragment.

[0066] In at least certain embodiments, the Disclosure provides nucleic acids encoding SIRPα-binding molecules according to the Disclosure. In at least certain embodiments, the Disclosure provides expression vectors comprising the nucleic acids of the Disclosure. In at least certain embodiments, the Disclosure provides cells comprising the expression vectors of the Disclosure. In at least certain embodiments, the Disclosure provides a method for producing SIRPα-binding molecules, the method comprising culturing cells or populations of such cells under conditions that promote the expression of SIRPα-binding molecules from the expression vectors by the cells. In various embodiments, the method for producing SIRPα-binding molecules provided herein further comprises isolating SIRPα-binding molecules from cells or populations of cells, or from a medium in which cells or populations of cells were cultured. In at least certain embodiments, the Disclosure provides a pharmaceutical composition comprising SIRPα-binding molecules of the Disclosure and pharmaceutically acceptable carriers or excipients.

[0067] In at least certain embodiments, the Disclosure provides a method for treating or delaying the progression of cancer in a subject, the method comprising administering an effective amount of the SIRPα-binding molecule, binding molecule, or pharmaceutical composition of the Disclosure to the subject, thereby treating and / or delaying the progression of cancer in the subject. In various embodiments, cancer is adenocarcinoma, cholangiocarcinoma (biliary tract cancer), bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, colon cancer, endometrial cancer, esophageal cancer, glioma, head and neck cancer, head and neck squamous cell carcinoma, leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer, kidney cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, or urothelial carcinoma. In various embodiments, the method further includes administering an additional therapeutic agent to treat cancer. In various embodiments, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint inhibitor. [Brief explanation of the drawing]

[0068] [Figure 1A] This document describes six anti-EGFR antibodies. The constructs included necitumumab (11F8)hIgG1 (C-1), necitumumab (11F8)hIgG1 EEF (C-2), cetuximab hIgG1 EEF (C-3), nimotuzumab hIgG1 EEF (C-4), P2X hIgG1 EEF (C-5), and 11F8 scFv hIgG1 EEF (C-6). The 11F8 scFv hIgG1 EEF construct (C-6) included the G44HC / G100LC scFv stabilizing mutation. EEF = Enhanced effector function (mediated by the G236A / S239D / I332E mutation in FC, represented by dots). [Figure 1B] The results obtained from flow cytometry assays to evaluate the binding of anti-EGFR antibodies to EGFR cells in A431 (high EGFR) and MCF7 (low EGFR) cells are shown. The results are presented as mean fluorescence intensity (MFI). [Figure 2A]This document describes four anti-EGFR-SIRPα fusion constructs containing the EGFR antibody necitumumab (11F8)hIgG 1EEF or 11F8 scFv IgG1 EEF and domain 1 of SIRPα (SIRPα-D1). The position of SIRPα varied between the constructs. For the 11F8 scFv IgG1 EEF-SIRPα fusion construct, SIRPα-D1 was fused to the C-terminus of the Fc (C-7). For the necitumumab (11F8)hIgG1 EEF-SIRPα fusion construct, SIRPα-D1 was fused to the C-terminus of the heavy chain (fusion 1, C-8), to the C-terminus of the light chain (fusion 2, C-9), or to the N-terminus of the light chain (fusion 3, C-10). The 11F8 scFv IgG1 EEF-SIRPα fusion construct (C-7) and C-terminal light chain fusion construct (C-9) included the G44HC / G100LC scFv stabilizing mutation. EEF = Enhanced effector function (mediated by the G236A / S239D / I332E mutation in FC, represented by dots). [Figure 2B] The results obtained from flow cytometry assays to evaluate CD47 cell binding of anti-EGFR-SIRPα fusion constructs to MCF7 WT cells (CD47+ / EGFR-) and MCF7 CD47 knockout cells (KO;CD47- / EGFR-) are shown. Results are presented as mean fluorescence intensity (MFI). Necitumumab hIgG1 EEF(C-2) and SIRPα-free 11F8 scFv hIgG1 EEF(C-6) were used as negative controls for CD47 binding. [Figure 3A] This diagram illustrates the anti-EGFR biparatopic VHH hIgG1 EEF SIRPα fusion construct (C-64) and the control construct without SIRPα fusion (C-63). EEF = enhanced effector function (mediated by the G236A / S239D / I332E mutation in FC, represented by dots). [Figure 3B]The results obtained from flow cytometry assays to evaluate CD47 cell binding of the anti-EGFR biparatopic VHH hIgG1 EEF SIRPα fusion (C-64) to MCF7 WT cells (CD47+ / EGFR-) and MCF7 CD47 knockout cells (KO;CD47- / EGFR-) are shown. Results are presented as mean fluorescence intensity (MFI). Necitumumab hIgG1 EEF (C-2) and the SIRPα-free anti-EGFR biparatopic VHH hIgG1 EEF construct (C-63) were used as negative controls for CD47 binding. [Figure 3C] The results obtained from flow cytometry assays to evaluate EGFR cell binding of the anti-EGFR biparatopic VHH hIgG1 EEF SIRPα fusion (C-64) to A431 WT (high EGFR / CD47+) and A431 CD47 KO (high EGFR / CD47-) cells are shown. Results are presented as mean fluorescence intensity (MFI). Necitumumab hIgG1 EEF (C-2) and SIRPα-free anti-EGFR biparatopic VHH hIgG1 EEF construct (C-63) were used as positive controls for EGFR binding. [Figure 4A] This diagram illustrates anti-EGFR-anti-CD47 fusion constructs containing the EGFR antibody necitumumab (11F8) hIgG1 EEF and anti-CD47 VHH antibody. The position of the anti-CD47 VHH antibody varied between constructs. The anti-CD47 VHH antibody was fused to the C-terminus of the heavy chain (fusion 1, C-11), to the C-terminus of the light chain (fusion 2, C-12), or to the N-terminus of the light chain (fusion 3, C-13). The C-terminal light chain fusion construct (C-12) included the G44HC / G100LC scFv stabilizing mutation. EEF = Enhanced effector function (mediated by the G236A / S239D / I332E mutation in FC, represented by dots). [Figure 4B]The results obtained from flow cytometry assays to evaluate the CD47 cell binding of the nesitumumab-anti-CD47 VHH fusion to MCF7 WT cells (CD47+ / EGFR-) and MCF7 CD47 knockout cells (KO;CD47- / EGFR-) are shown. Results are presented as mean fluorescence intensity (MFI). Necitumumab hIgG1 EEF(C-2) was used as a negative control for CD47 binding. [Figure 5A] This document describes an exemplary workflow for phagocytic screening of multispecific antigen constructs. THP-1 monocytes stably expressing the nuclear-only tag GFP2 were used as effector cells, and A431 human epidermal carcinoma cells labeled with pHrodo Orange were used as target cells. Cells were incubated with the multispecific antigen constructs and imaged every 45 minutes for 12 hours using phase, green, and orange channels. Since pHrodo fluoresces only at low pH (phagocytosis), phagocytosed cells were defined as a green + high-intensity orange overlap. [Figure 5B] The amount of phagocytosis after adding each fusion construct (C-1 to C-15 and C-64 to C-64) at a concentration of 50 nM is shown. The results are shown as the area under the curve (AUC). [Figure 5C] The amount of phagocytosis after addition of necitumumab (11F8)hIgG1 EEF SIRPα fusion constructs (C-8, C-9, and C-10) at concentrations of 0.5, 5, and 50 nM is shown. Results are presented as area under the curve (AUC). Necitumumab (11F8)hIgG1 (C1) and necitumumab (11F8)hIgG1 EEF (C2) constructs were used as non-SIRPα controls. A control lacking the anti-EGFR domain (C-14, CD47 blocker only), in which SIRPα was fused to the C-terminus of the homodimer Fc IgG1 EFF polypeptide, was also used alone or in combination with necitumumab (11F8)hIgG1 EEF (C2, anti-EGFR only). [Figure 5D]This shows the amount of phagocytosis after adding a 0.5 nM construct consisting of a CD47 blocker only (C14, Fc IgG1 EFF-SIRPα), a tumor targeting only construct (nesitumumab (11F8) hIgG1 EEF; C2), a combination of a CD47 blocker (C14) construct and an anti-EGFR (C2) construct, or a multispecific fusion construct (nesitumumab (11F8) hIgG1 EEF SIRPα fusion 1; C-8). The results are expressed as a change in phagocytosis ratio. An asterisk indicates P < 0.0001, which was determined from a two-way ANOVA with Tukey multiple comparison test. [Figure 6A] This describes a construct consisting solely of a CD47 blocker (without anti-EGFR), in which SIRPαD1 is fused to either the C-terminus (SIRPα D1 Fc fusion 1, C-14) or N-terminus (SIRPα D1 Fc fusion 2, C-15) of a homodimer Fc IgG1 EFF polypeptide. A construct using the extracellular domain of CD47 as a "shielding domain" to block the interaction between SIRPα-D1 and cell surface CD47 is also shown. The extracellular domain of CD47 and SIRPα-D1 are each ligated to one of the polypeptide chains of a heterodimer Fc having a knob-in-hole mutation, at either the N-terminus (KiH Fc EEF CD47 and SIRPα fusion 1, C-16M) or C-terminus (KiH Fc EEF CD47 and SIRPα fusion 2, C-17M) of each Fc polypeptide. The interaction between the CD47 extracellular domain and SIRPα D1 was conditionally controlled by linking the shielding domain to the Fc region using a cleavable linker. [Figure 6B] The results of flow cytometry assays to evaluate the CD47 cell binding of SIRPαD1-Fc fusions (C14, C15, C16M, and C17M) with or without shielding domains to MCF7 WT (CD47+ / EGFR-) cells, MCF7 KO (CD47- / EGFR+) cells, A431 WT (CD47+ / EGFR+) cells, and A431 KO (CD47+ / EGFR+) cells are shown. Results are presented as mean fluorescence intensity (MFI). [Figure 7A] Three forms of the anti-EGFR-SIRPα shielding fusion construct are described. The forms include nesitumumab(11F8)hIgG1 EEF:SIRPα-WT:shielding fusion (formula number 1), nesitumumab(11F8)hIgG1 EEF:SIRPα-CV1:shielding fusion (formula number 2), and the asymmetric variant KiH nesitumumab(11F8)hIgG1 EEF (where the whole arm is fused with SIRPα-CV1 and the knob arm is fused with the shielding) (formula number 3). [Figure 7B] The results obtained from flow cytometry assays to evaluate the CD47 cell binding of anti-EGFR-SIRPα-shielding fusion constructs to MCF7 WT (CD47+ / EGFR-) cells and MCF7 KO (CD47- / EGFR+) cells are shown. Fusion constructs were assayed with or without digestion by matryptase at concentrations of 100 nM to 0.6 pM. Exemplary results for the following constructs are shown: unshielded (C-50), shone with protease (C-55M digestion), shone with (C-55M undigested), and unshielded with an uncleavable linker (C61). Results are shown as median fluorescence intensity (MFI) plotted against the concentration of the fusion construct. [Modes for carrying out the invention]

[0069] Detailed explanation This disclosure relates to compositions and methods useful for treating diseases (e.g., cancer). Specifically, this disclosure provides compositions that can selectively direct myeloid cell activity (e.g., direct and / or indirect death by myeloid cells directed toward target cells) to target cells, such as by selective activation in specific target conditions (e.g., an activated tumor microenvironment). Among the embodiments provided are multispecific binding molecule platforms that are selectively activated in tumors to promote tumor cell death by macrophages. The platforms include various configurations that combine targeting tumors, pre-preparing with a shielding domain for conditional activation to block antiphagocytic protein activity in a tumor-selective manner, and promoting tumor cell death by promoting macrophage-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP). In some embodiments, the therapeutic conjugates encompassed by this disclosure can selectively lead to the elimination of target cells by antibody-dependent cell-mediated phagocytosis (ADCP). In some embodiments, the therapeutic conjugates encompassed by this disclosure can selectively lead to the elimination of target cells by antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the therapeutic conjugates encompassed by this disclosure can selectively lead to the elimination of target cells by direct death.

[0070] There is a need for improved therapeutics that can enhance the potential phagocytic activity of myeloid cells, including macrophages. Macrophages make up up to 50% of all cells in solid tumors (Mellman et al, Immunity (2023), Cheng et al. J. Hematology & Oncology (2023)). However, existing therapies are not yet effective in utilizing macrophages for direct tumor death. For example, the immunosuppressive tumor microenvironment created by macrophages is a major obstacle to the success of immuno-oncology. Although macrophages have the ability to phagocytose and kill tumor cells, they are often inactivated and immunosuppressive in the tumor microenvironment. For example, many tumor cells and cells in the tumor microenvironment express CD47, which suppresses the phagocytic activity of macrophages. Therefore, the therapeutic potential of macrophages, including their potential to promote rapid innate immune-driven tumor debulking and to bridge the gap between innate and adaptive immune responses to prolong clinical responses, is not fully realized. Therefore, there is a great need to release macrophages to give direct tumor elimination, including as part of an organized immune attack that can achieve a durable response.

[0071] In some embodiments, the therapeutic conjugates encompassed by this disclosure may include (1) at least one binding domain (APP binding domain) that binds to an antiphagocytic protein (APP) (e.g., expressed on myeloid cells or on cells targeted for regulation (e.g., direct and / or indirect death)); (2) at least one binding domain (shielding domain) that conditionally inhibits the interaction between the anti-APP binding domain and its target; and (3) a proteolytically cleavable linker, the cleavage of which removes the inhibition of APP binding by the shielding domain, thereby achieving activatable activity. The therapeutic conjugates encompassed by this disclosure may include one or more additional elements (e.g., one or more elements that promote and / or activate myeloid cells) and / or at least one binding domain (target cell binding domain) that binds to a target cell antigen. For example, the conjugates provided are multispecific conjugates further comprising an immunoglobulin Fc region for activating FcγR. In some embodiments, the immunoglobulin Fc region includes Fc-enhancing mutations that are further known to enhance macrophage activity and promote macrophage-dependent ADCC / ADCP. Fc-enhancing mutations include those that increase affinity for FcγRIIIa compared to the inhibitory receptor FcγRIIb (e.g., S239D and I332E) and those that increase affinity for FcγRIIa (e.g., G236A). For example, the Fc-enhancing triple mutations S239D, I332E, and G236A can improve the ratio of RIIa binding to RIIb binding while maintaining increased affinity for FcγRIIIa. These elements, therapeutic conjugates containing one or more such elements, and their uses (e.g., for the treatment of cancer) are further described herein.

[0072] In the embodiments of the provided conjugate, APP is CD47, and the conjugate comprises at least one binding domain for binding to CD47 and blocking the suppression of macrophages and other myeloid cells. Blocking CD47 can induce long-term antitumor immunity and can also bridge the innate and adaptive immune systems. Furthermore, blocking CD47 can be combined with a conjugate that enables FcγR interaction to further enhance myeloid-driven antitumor responses (including those against distal tumors). In addition, the provided conjugate is conditionally activated to specifically control binding to APP in the tumor microenvironment rather than in tumor tissue by using a shielding domain that can be released in the tumor, thereby maximizing selective activation in the tumor and specifically promoting tumor cell death by macrophages without targeting normal cells. For example, in the embodiments provided, tumor selectivity can be achieved by protease-mediated activation of a proteolytically cleavable linker that links the shielding domain to the APP binding domain, thereby selectively activating the provided conjugate in the tumor. This addresses the shortcomings of some existing CD47 blockade therapies, given the ubiquitous presence of CD47 in both normal and tumor cells. Off-tumor binding to CD47 can result in toxicity and poor pharmacological action (including anemia, thrombocytopenia, and reduced tissue absorption (availability)), while tumor-specific on-target binding is expected to yield antitumor activity and promote ADCC / ADCP, thereby accelerating tumor cell death. The proposed approach also offers flexibility by allowing modification by using a SIRPα binding domain as the binding domain to CD47 APP, including wild-type SIRPα and various engineered variants with moderate to high affinity. The proposed approach provides a unique therapeutic approach that enables enhancement of Fc function and CD47 blockade without other sacrifices (such as silencing Fc or using low-affinity CD47 blockers).

[0073] This disclosure further relates to functional equivalents of APP-binding domains (i.e., APP-binding domain means), shielding domains (i.e., shielding domain means), proteolytically cleavable linkers (i.e., proteolytically cleavable linker means), myeloid-promoting and / or activating elements (i.e., myeloid-promoting means and / or myeloid-activating means), and / or target cell-binding domains (i.e., target cell-binding domain means). Each functional equivalent of each element performs substantially the same function as the respective element using substantially the same method to obtain substantially the same results, and is therefore substantially not different from the respective elements described herein. Each functional equivalent of each element can be prepared and / or screened by well-known methods (such as assays for analyzing binding, affinity, phagocytosis (e.g., ADCP), cell death (e.g., ADCC or CDC), cytokine release, immune cell activation, immune cell proliferation, and / or immune cell migration), and representative examples of such functions, elements, and / or equivalents are further described herein.

[0074] This disclosure is at least in part based on the recognition that cells associated with certain diseases, conditions, or disorders (these terms may be used interchangeably herein), such as cancer, may contribute to the pathogenesis by evading phagocytosis through abnormal and / or increased expression of APP. Increased expression of APP (e.g., CD47) inhibits phagocytosis and correlates with poor prognosis. The conjugates provided herein conditionally (activatably) direct myeloid cell activity to such cells, thereby treating the disease.

[0075] All publications, including patent documents, scientific papers, and databases, referenced in this application are incorporated by reference in their entirety for any purpose to the same extent as each individual publication is incorporated by reference individually. If any definitions contained herein conflict with or contradict any definitions contained herein by reference in patents, applications, publications, and other publications incorporated herein by reference, the definitions contained herein shall prevail.

[0076] Section headings used in this specification are for structural purposes only and should not be interpreted as limiting the subjects described herein.

[0077] I. Definition A, An, The, Or: As used herein, “a,” “an,” and “the” refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, “an element” discloses embodiments of strictly one element and embodiments comprising multiple elements. As used herein, the terms “or” and “and / or” as conjunctions in a list of at least two elements encompass and disclose embodiments in which the listed elements are included alternately, together, or in any combination.

[0078] Approximate: In some embodiments, the term "approximately" encompasses values ​​(including extreme values) within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the measured value, or any range between them (e.g., ±2% to 6%). In some embodiments, the term "approximately" refers to error variability inherent in the method, assay, or measurement (such as variability between experiments).

[0079] Affinity: As used herein, "affinity" refers to the total strength of non-covalent interactions between a specific binding agent (e.g., an antigen binding agent) and / or its binding site and a binding target (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the 1:1 interaction between a binding agent and its binding target (e.g., an antibody and the antigen target of the antibody). Those skilled in the art will understand that changes in affinity can be described by comparison to a reference (e.g., an increase or decrease when compared to a reference) or can be described numerically. Affinity can be measured and / or expressed by a number of methods known in the art, including but not limited to equilibrium dissociation constant (K D ) and / or equilibrium binding constant (K A ). K D is k off / k on , while K A is k on / k off , k on refers to the association rate constant (e.g., the association rate constant for the binding of an antibody and an antigen), and k off refers to dissociation (e.g., dissociation of an antibody from an antigen). k on and k off can be determined by techniques known to those skilled in the art, such as BIACORE® or KinExA.

[0080] Agent: As used herein, the term "agent" can refer to any chemical entity, including but not limited to any one or more of atoms, molecules, compounds, amino acids, polypeptides, nucleotides, nucleic acids, proteins, protein complexes, liquids, solutions, sugars, polysaccharides, lipids, or combinations or complexes thereof.

[0081] Antibodies: As used herein, the term “antibody” refers to a polypeptide comprising one or more standard immunoglobulin sequence elements (e.g., heavy chain variable domains, light chain variable domains, and / or one or more CDRs) sufficient to confer specific binding to a particular antigen. Therefore, the term “antibody” includes, but is not limited to, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents containing them. Antibodies may be naturally occurring immunoglobulins (e.g., those produced by organisms that react to antigens). Synthetic antibodies, non-naturally occurring antibodies, or engineered antibodies can be obtained by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those skilled in the art.

[0082] As is well known in the art, a typical human immunoglobulin is a tetramer of approximately 150 kD containing two identical heavy (H) chain polypeptides (approximately 50 kD each) and two identical light (L) chain polypeptides (approximately 25 kD each) that are linked together to form a structure commonly referred to as a "Y-shaped" structure. Typically, each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain contains three CH domains: CH1, CH2, and CH3. A short region known as a "switch" connects the heavy chain variable domain and the heavy chain constant domain. A "hinge" connects the CH2 and CH3 domains to the remainder of the immunoglobulin. Each light chain contains a light chain variable domain (VL) and a light chain constant domain (CL), which are separated from each other by another "switch". Each variable domain contains three hypervariable loops (CDR1, CDR2, and CDR3) known as "complementarity-determining regions," as well as four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs are arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and / or light chains are typically understood to provide binding sites that can interact with antigens. The constant domains can mediate the binding of antibodies to various immune system cells (e.g., effector cells and / or cytotoxicity-mediating cells), receptors, and elements of the complement system. The heavy and light chains are linked to each other by a single disulfide bond, and the other two disulfide bonds connect the heavy chain hinge regions to each other, resulting in the dimers being linked to each other and forming a tetramer. When natural immunoglobulins fold, the FR region forms a beta sheet that gives the domain a structural framework, and the CDR loop regions, derived from both the heavy and light chains, come together in three-dimensional space, resulting in the creation of a single hypervariable antigen-binding site located at the tip of the Y structure.

[0083] In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a monospecific antibody, or a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the antibody comprises at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer comprising two heavy chain monomers and two light chain monomers. Furthermore, the term “antibody” may include, but is not limited to, any construct or form known in the art that utilizes antibody structure and / or functional properties (unless otherwise stated or evident from the context), intrabodies, domain antibodies, antibody mimes, Zybody®, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, isolated CDRs or sets thereof, single-chain antibodies, single-chain Fv(scFv), disulfide-linked Fv(sdFv), polypeptide-Fc fusions, single-domain antibodies (e.g., shark single-domain antibodies (IgNAR, etc.) or fragments thereof), camel antibodies, camelized antibodies, shielding antibodies (e.g., Probody®), afibody, anti-idiot antibodies This includes (anti-Id) antibodies (e.g., anti-anti-Id antibodies), single-chain or tandem diabolic bodies (TandAb®), VHH, Anticalin®, Nanobody® minibodies, BiTE®, Ankyrin repeat proteins or DARPIN®, Avimer®, DART, TCR-like antibodies, Adnectin®, Affilin®, Trans-body®, Affibody®, TrimerX®, Microprotein, Fynomer®, Centyrin®, KALBITOR®, CAR, engineered TCRs, and antigen-binding fragments of any of the above.

[0084] Single-domain antibody: As used herein, the term “single-domain antibody” may be interchangeable with “VHH domain” or “heavy-chain-only antibody variable domain” and refers to a single-chain antigen-binding domain that has the ability to bind to an antigen or epitope independently of a second light-chain variable domain. Single-domain antibodies (VHH) are distinguished from variable domains derived from the heavy-chain variable domain present in conventional quadruple-chain antibodies (referred to herein as “VH domain” or “VH region”) and from variable domains derived from the light-chain variable domain of conventional quadruple-chain antibodies (referred to herein as “VL domain” or “VL domain”). While VHH domains may be human domains, VHH domains also include single domains derived from other species, such as rodent, nurse shark, and camel VHH domains. Camel VHH is an immunoglobulin single-variable-domain polypeptide derived from species that produce heavy-chain antibodies that naturally lack a light chain (including camels, llamas, alpacas, dromedaries, and guanacos). Such VHH domains can be humanized according to standard techniques available in the art and are considered “single-domain antibodies.” VHH includes camel VHH domains (including those derived from species including camels, llamas, alpacas, dromedaries, and guanacos) and humanized VHH domains. Such VHH domains can be humanized according to standard techniques available in the art. VHH domains also include synthetic VHH domains, such as human-like VHH domains (where the amino acids at positions 44 and 45 or 37, 44, 45, and 47, based on Kabat numbering, each contain the amino acids at the corresponding positions in camel VHH (see, for example, PCT Publication WO2021 / 178263)).

[0085] A “humanized” antibody refers to an antibody (e.g., VHH) that contains amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, all or substantially all of the CDRs of a humanized VHH correspond to those of a non-human VHH, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody.

[0086] Affinity-matured antibodies (such as VHH) have one or more modifications in one or more CDRs, resulting in improved affinity for the antigen compared to their respective parental albumin-binding molecules. The affinity-matured albumin-binding molecules of the present invention can be prepared by methods known in the art (e.g., as described in KS Johnson and RE Hawkins, “Affinity maturation of antibodies using phage display”, Oxford University Press 1996).

[0087] In some embodiments, the antibody comprises one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs) or variable domains. In some embodiments, the antibody may be a covalently modified ("complexed") antibody (for example, an antibody comprising a polypeptide containing one or more standard immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, wherein the polypeptide is covalently linked to one or more of the following: a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, the antibody sequence elements may be humanized, primated, chimeric, etc., as is known in the art.

[0088] Antibodies containing a heavy chain constant domain may be antibodies of any known class (including, but not limited to, IgA, secreted IgA, IgG, IgE, and IgM) based on the heavy chain constant domain amino acid sequence (e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ)). IgG subclasses are also well known to those skilled in the art, and include, but not limited to, human IgG1, IgG2, IgG3, and IgG4. “Isotype” refers to an Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant domain gene. As used herein, “light chain” may be of a distinct type (e.g., kappa (κ) or lambda (λ)) based on the amino acid sequence of the light chain constant domain. In some embodiments, the antibody has a constant domain sequence characteristic of mouse, rabbit, primate, or human immunoglobulin. Naturally produced immunoglobulins are glycosylated, typically the CH2 domain being glycosylated. As is known in the art, the affinity and / or other binding properties of an Fc domain (which can be interchangeably referred to as the “Fc region”) to an Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, antibodies may lack covalent modifications (e.g., glycan addition) that they would have if naturally produced. In some embodiments, antibodies produced and / or utilized according to this disclosure include a glycosylated Fc domain (including Fc domains in which such glycosylation has been modified or manipulated).

[0089] Antibody-dependent cell-mediated cytotoxicity (ADCC): This term refers to a form of cytotoxicity in which secreted antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to target cells containing an antigen, and subsequently kill the target cells. To evaluate the ADCC activity of a target molecule, an in vitro ADCC assay (such as those described in U.S. Patent No. 5,500,362 or 5,821,337) may be performed. As is well known in the art, the Fc moiety can be manipulated to produce a desired interaction with or lack thereof with the Fc receptor.

[0090] Antibody fragment: As used herein, “antibody fragment” refers to an antibody fragment or antibody preparation described herein, typically the portion containing an antigen-binding moiety or its variable region. Antibody fragments can be produced by any means. For example, in some embodiments, antibody fragments can be produced enzymatically or chemically by fragmentation of an intact antibody or antibody preparation. Alternatively, in some embodiments, antibody fragments can be produced recombinantly (i.e., by the expression of an engineered nucleic acid sequence). In some embodiments, antibody fragments can be produced entirely or partially by synthesis. In some embodiments, antibody fragments (specifically, antigen-binding antibody fragments) may have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, and in some embodiments, may have a length of at least about 200 amino acids.

[0091] Antigen-binding domain: The term “antigen-binding domain” or “binding region” refers to the portion of a binding molecule (such as an immunoglobulin molecule) that is involved in antigen binding. For conventional quadruple-chain antibodies, or for Fab fragments, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments), or diabodies, or other antibody fragments derived from conventional quadruple-chain antibodies, the antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. In these cases, binding to each epitope of the antigen occurs by a pair (bond) of immunoglobulin domains (such as the light chain variable domain and the heavy chain variable domain), i.e., by the VH-VL pair of immunoglobulin domains that bind together to each antigen epitope. Three highly variable segments within the V regions of the heavy and light chains, called the “hypervariable region,” are inserted between more conserved adjacent segments, known as the “framework region” or “FR.” Therefore, the term "FR" refers to the naturally occurring amino acid sequences between and adjacent to the hypervariable regions in immunoglobulins. In antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in relation to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen to which it is bound, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs." The assignment of amino acids to each domain follows the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989). For single-domain antibodies or VHHs, the antigen-binding domain contains a heavy chain variable domain which includes three CDRs of the heavy chain variable domain.The VHH domain can specifically bind to an epitope without an additional antigen-binding domain (in contrast to the VH or VL domain of conventional quadruple-chain antibodies, in which case the epitope is recognized by the VL domain combined with the VH domain). Many proteins also contain an immunoglobulin domain (known as an immunoglobulin-like (Ig-like) domain), which is a protein region homologous to the V or C domain of the immunoglobulin protein responsible for binding to the antigen in immunoglobulins. These immunoglobulin domains in proteins of the immunoglobulin superfamily are classified as IgV domains or IgC domains and can function as antigen-binding domains involved in antigen binding. Linked: Two events or entities are “linked” to each other if, as the term is used herein, the presence, level, and / or form of one correlates with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microorganism, etc.) is considered associated with a particular disease, disorder, or condition if its presence, level, and / or form correlate (e.g., across relevant populations) with the incidence and / or susceptibility to that disease, disorder, or condition. In some embodiments, two or more entities are physically “linked” to one another if they interact directly or indirectly, and as a result, they are and / or remain physically close to one another. In some embodiments, two or more physically linked entities are covalently bonded to one another, and in some embodiments, two or more physically linked entities are not covalently bonded to one another but are non-covalently bonded, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, or a combination thereof.

[0092] Bonding: As used herein, the term “bonding” refers to a non-covalent bond between or between two or more agents. A “direct” bond involves physical contact between agents, while an indirect bond involves physical interaction through physical contact with one or more intermediate agents. Bonding between two or more agents can occur and / or be evaluated in any of a variety of situations, including when the interacting agents are studied individually or in relation to more complex systems (e.g., covalently bonded to a carrier agent or otherwise, and / or in biological systems or cells).

[0093] The term "specific binding" or "specifically binding" refers to a type of non-covalent interaction that occurs between binding partners (such as between a receptor and its ligand, or between an immunoglobulin molecule and an antigen), where the binding interaction is specific or high-affinity. The strength or affinity of an immunological binding interaction is expressed by the dissociation constant (K) of the interaction. D ) can be expressed in terms of K DA smaller value indicates greater affinity. The immunological binding characteristics of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on geometric parameters that equally affect the concentration of the complex partner, the affinity of the interaction, and the rates in both directions. Thus, both the "on-rate constant" (Kon) and the "off-rate constant" (Koff) can be determined by calculating the concentration and the actual binding and dissociation rates. (See Nature 361:186-87 (1993)). The Koff / Kon ratio allows for the cancellation of all parameters unrelated to affinity and corresponds to the dissociation constant Kd. (See Davies et al. (1990) Annual Rev Biochem 59:439-473 in general). The antibodies of this disclosure are said to bind specifically to an antigen (e.g., EGFR) when measured by an assay (such as a radioligand binding assay or a similar assay known to those skilled in the art), the binding constant (Kd) is ≤1 μM, for example, ≤100 nM in some embodiments, ≤10 nM in some embodiments, and ≤100 pM to about 1 pM in some embodiments.

[0094] A protein (such as a protein, immunoglobulin, antibody, or immunoglobulin monovariate) that is capable of “binding” or “specifically binding” to a particular epitope, antigen, or protein, and that has “affinity” and / or “specificity” to it, is said to be “targeted” or “directed” to such epitope, antigen, or protein, or is a “binding” molecule to such epitope, antigen, or protein.

[0095] Cancer: As used herein, the term “cancer” means a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in an abnormal growth phenotype characterized by an abnormally increased growth rate and / or a marked loss of control over cell proliferation. In some embodiments, cancer may comprise one or more tumors. In some embodiments, cancer may comprise or comprise precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or nonmetastatic cells. In some embodiments, cancer may comprise or comprise a solid tumor. In some embodiments, cancer may comprise or comprise a hematological tumor.

[0096] Chemotherapy Agents: As used herein, the term “chemotherapy agent” means one or more agents known to treat or contribute to the treatment of cancer, or having characteristics known to do so, in accordance with their use in the art. Specifically, chemotherapy agents include apoptosis promoters, cell growth inhibitors, and / or cytotoxic agents. In some embodiments, chemotherapy agents may be alkylating agents, anthracyclines, cytoskeletal disruptors (e.g., microtubule-targeting moieties such as taxanes, mytansines, and their analogues), epothyrons, histone deacetylase (HDAC) inhibitors, topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), kinase inhibitors, nucleotide analogues or nucleotide precursor analogues, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids, and / or analogues sharing related antiproliferative activity. In some specific embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auiristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epotilon, etoposide, fluorouracil, gemcitabine The chemotherapeutic agent may be vin, hydroxyurea, idarubicin, imatinib, irinotecan, mytansine and / or its analogues (e.g., DM1), mechloretamine, mercaptopurine, methotrexate, mitoxantrone, mytansinoid, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, barrubicin, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. In some embodiments, the chemotherapeutic agent may be used in association with an antibody-drug conjugate.In some embodiments, the chemotherapeutic agent is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-dox Sorbicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glenbatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA These are found in antibody-drug conjugates selected from ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mahodotin, and lorbotuzumab meltansine. In some embodiments, the chemotherapeutic agent is or may include farnesyl thiosalicylic acid (FTS), 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2), tetramethoxystilbene (TMS), δ-tocatrienol, sarinomycin, or curcumin.

[0097] Complement-dependent cell injury (CDC): This term refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to an antibody bound to a corresponding antigen. A CDC assay (e.g., as described in Gazzano-Santoro et al. (1997)) can be performed to assess complement activation.

[0098] Domain: As used herein, the term “domain” refers to a section or portion of an entity. In some embodiments, a “domain” is associated with certain structural and / or functional properties of an entity such that, if the domain were physically separated from the rest of its parent entity, the domain would substantially or completely retain certain structural and / or functional properties. Alternatively or additionally, a domain may be a portion of an entity, or include it, that, if separated from its (parent) entity and conjugated to a different (recipient) entity, substantially retain and / or confer to the recipient entity one or more structural and / or functional properties that characterized it in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide, and in some such embodiments, the domain is characterized by specific structural elements (e.g., specific amino acid sequences or sequence motifs, α-helix features, β-sheet features, coiled-coil features, random-coil features, etc.) and / or specific functional properties (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.). In some embodiments, the domain is a characteristic moiety or characteristic sequence element, or comprises such a moiety or sequence element.

[0099] Environment or Microenvironment: The terms “environment” and “microenvironment” generally refer to a localized area or feature within a tissue region of interest, for example, “tumor microenvironment.” The term “tumor microenvironment” or “TME” refers to the surrounding microenvironment that constantly interacts with tumor cells, facilitating crosstalk between tumor cells and their environment. The tumor microenvironment may include the tumor’s cellular environment, surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix. The tumor environment may include tumor cells or malignant cells that are supported and influenced by the tumor microenvironment to ensure their growth and survival. The tumor microenvironment may also include tumor-infiltrating immune cells (such as lymphoid and myeloid cells) and stromal cells (such as tumor-associated fibroblasts and endothelial cells that contribute to the structural integrity of the tumor) that can stimulate or inhibit the anti-tumor immune response. Stromal cells may include cells that contribute to structural integrity (fibroblasts), such as endothelial cells and pericytes that constitute tumor-associated blood vessels, as well as tumor-associated macrophages (TAMs) and infiltrating immune cells (including monocytes, neutrophils (PMNs), dendritic cells (DCs), T cells and B cells, mast cells, and natural killer (NK) cells). While stromal cells make up the majority of tumor cells, macrophages are the dominant cell type in solid tumors.

[0100] Fragment: As used herein, “fragment” refers to a structure that is or contains a distinct portion of a reference agent (sometimes referred to as the “parent” agent). In some embodiments, a fragment is missing one or more portions found in the reference agent. In some embodiments, a fragment is one or more portions found in the reference agent, or contains them. In some embodiments, the reference agent is a molecule (such as a small molecule or other chemical entity). In some embodiments, a molecular fragment is at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the atoms and / or bonds found in the reference molecule.

[0101] In some embodiments, the reference agent is a polymer (such as a polynucleotide or polypeptide). In some embodiments, the polymer fragments are at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 The monomers consist of 1, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomer units (e.g., residues), or contain such units. In some embodiments, the polymer fragments represent or include at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomer units (e.g., residues) found in the reference polymer. The reference polymer fragments are not necessarily identical to the corresponding portions of the reference polymer. For example, a fragment of a reference polymer may be a polymer having a sequence of residues that has at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with the reference polymer. The fragment may or may not be produced by the physical fragmentation of the reference agent. In some examples, the fragment is produced by the physical fragmentation of the reference agent. In some examples, the fragment is not produced by the physical fragmentation of the reference agent, but can instead be produced by, for example, de novo synthesis or other means.

[0102] To improve, increase, inhibit, decrease, or reduce: As used herein, the terms “improve,” “increase,” “inhibit,” “decrease,” and “reduce,” as well as their grammatical equivalents, indicate a qualitative or quantitative difference from the reference.

[0103] Inhibit or Downcontrol: The terms “inhibit” or “downcontrol” include, for example, reducing, limiting, or blocking a particular action, function, or interaction. In some embodiments, cancer is “inhibited” if at least one symptom of cancer is reduced, terminated, slowed, or blocked. As used herein, cancer is also “inhibited” if cancer recurrence or metastasis is reduced, slowed, delayed, or blocked. Similarly, a biological function (such as the function of a protein) is inhibited if it is reduced compared to a reference state (such as a control, like the wild-type state). Such inhibition or deficiency may be induced, for example, by the application of an agent at a particular time and / or place, or it may be constitutive, for example, by a hereditary mutation. Such inhibition or deficiency may be partial or complete (e.g., essentially no measurable activity compared to a reference state, such as a control, like the wild-type state). In some embodiments, essentially complete inhibition or deficiency is referred to as “blocked.” In one embodiment, the term refers to reducing the level of a given output or parameter (e.g., background staining, biomarker signaling, biomarker immunoinhibitory function, and similar) to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or less than the amount in the corresponding control. Reducing the level of a given output or parameter may, but is not required, mean the absolute absence of the output or parameter. This disclosure does not require, but is not limited to, methods for the complete elimination of the output or parameter. A given output or parameter may be determined using methods well known in the art, as discussed herein, including, but not limited to, immunohistochemical, molecular biological, cell biological, clinical, and biochemical assays. The terms “facilitate” and “upregulate” have opposite meanings.

[0104] Linker: As used herein, “linker” is used to refer to a portion of a multi-element compound that connects different elements to one another. For example, a polypeptide having a structure containing two or more functional or organizational domains will often contain a sequence of amino acids between such domains that links them to one another, as will be understood by those skilled in the art. In some embodiments, a polypeptide containing a linker element has an overall structure in the general form S1-L-S2, where in the structure, S1 and S2 may be the same or different, representing two domains linked to one another by a linker. In some embodiments, the polypeptide linker has an amino acid length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more. In some embodiments, the linker does not take on a rigid three-dimensional structure, but rather tends to impart flexibility to the polypeptide. When manipulating polypeptides known in the art (e.g., fusion polypeptides), various different linker elements can be appropriately used (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448 and Poljak, RJ, et al. (1994) Structure 2:1 121-1123).

[0105] To adjust: The term "to adjust" and its grammatical equivalents refer to and encompass both increasing and decreasing, or both.

[0106] Functionally linked: As used herein, “functionally linked” means that at least one element and one element are linked together, resulting in a relationship that allows those component elements to function in their intended manner. For example, a nucleic acid sequence or amino acid sequence is functionally linked to another sequence if it modifies the expression, structure, or activity of the linked sequence (e.g., in its initial manner). Often, two nucleic acid sequences are functionally linked if they contribute to the expression, structure, or activity of a gene or encoded polypeptide. Often, two amino acid sequences are functionally linked if they are expressed as a single polypeptide.

[0107] Pharmacopoeia-acceptable: As used herein, the term “pharmacopoeia-acceptable” means that, when applied to one or more components for the formulation of a composition disclosed herein, each component must be compatible with the other components of the composition and harmless to its recipient.

[0108] pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium (such as a liquid or solid filler, diluent, excipient, or solvent encapsulant) that facilitates the formulation of a drug (pharmaceutical agent), modifies the bioavailability of a drug, or facilitates the transport of a drug from one organ or part of a subject to another. Some examples of materials that can act as pharmaceutically acceptable carriers include sugars (such as lactose, glucose, and sucrose); starches (such as corn starch and potato starch); cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate); powdered tragacanth; malt; gelatin; talc; excipients (such as cocoa butter and suppository waxes); oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil); glycols (such as propylene glycol); polyols (such as glycerin, sorbitol, mannitol, and polyethylene glycol); esters (such as ethyl oleate and ethyl laurate); agar; buffers (such as magnesium hydroxide and aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffers; polyesters, polycarbonates, and / or polyacid anhydrides; and other non-toxic suitable substances used in pharmaceutical formulations.

[0109] Pharmaceutical composition or formulation: As used herein, the terms “pharmaceutical composition” or “formulation” refer to a composition in which a therapeutic agent is formulated together with one or more pharmaceutically acceptable carriers.

[0110] Reference: As used herein, “reference” refers to the standard or control on which the comparison is made. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual, or population thereof, or a measure or feature representing it, is compared to the agent, sample, sequence, subject, animal, or individual, or population thereof, or a measure or feature representing it, which is the reference. In some embodiments, the reference is a measured value. In some embodiments, the reference is an established standard or expected value. In some embodiments, the reference is a historical reference. The reference may be quantitative or qualitative. Typically, as a person skilled in the art would understand, the reference and the value it is compared to correspond to an evaluation under equivalent conditions. A person skilled in the art would understand under what circumstances sufficient similarity exists to justify reliability and / or comparison. In some embodiments, a suitable reference may be an agent, sample, sequence, subject, animal, or individual, or population thereof, or a measure or feature representing it, under conditions that a person skilled in the art would recognize as equivalent (for example, for the purpose of evaluating one or more specific variable elements, e.g., the presence or absence of an agent or state).

[0111] Small molecules: The term “small molecules” is a term used in the art and includes molecules with a molecular weight of less than approximately 1000 or less than approximately 500. In one embodiment, small molecules do not necessarily consist solely of peptide bonds. In another embodiment, small molecules are not oligomers. Exemplary small molecule compounds that may be screened for activity include, but are not limited to, peptides, peptide mimes, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compound is a small organic non-peptide compound. Unless otherwise specified, the term is intended to encompass all stereoisomers, geometric isomers, tautomers, and isotopes of the chemical structure of interest.

[0112] Therapeutic Agent: As used herein, the term “therapeutic agent” refers to any agent that, when administered to a subject, induces a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across a suitable population. In some embodiments, a suitable population may be a model organism (e.g., an animal model of a disorder of interest (such as cancer), a humanized animal model, an animal model including the human immune system, and similar) or a human population. In some embodiments, a suitable population may be defined by various criteria (e.g., a particular age group, sex, genetic background, pre-existing clinical condition). In some embodiments, a therapeutic agent is a substance that can be used to treat a disease, disorder, or condition. In some embodiments, a therapeutic agent is an agent that has been or needs to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent that requires a medical prescription for administration to humans.

[0113] Subject: As used herein, the term “subject” means an organism, typically a mammal (e.g., human, rat, or mouse). In certain embodiments, the subject is human. In some embodiments, the subject suffers from a disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not suffer from a disease, disorder, or condition. In some embodiments, the subject exhibits no symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject has one or more characteristics characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, the subject is a subject being tested for and / or administered treatment for a disease, disorder, or condition. In some cases, a human subject may be interchangeably referred to as “patient” or “individual.” A subject administered with an agent associated with a treatment for a disease, disorder, or condition associated with the subject may be referred to as a subject requiring the agent, i.e., a subject needing it.

[0114] Therapeutic dose: As used herein, “therapeutic dose” means a dose that produces the desired effect for which it is intended to be administered. In some embodiments, the term means a dose sufficient to treat a disease, disorder, and / or condition when administered according to a therapeutic drug regimen to a population that is suffering from or susceptible to the disease, disorder, and / or condition. In some embodiments, a therapeutic dose is one that reduces the incidence and / or severity of one or more symptoms of the disease, disorder, and / or condition, and / or delays their onset. Those skilled in the art will understand that a therapeutic dose does not necessarily guarantee success of treatment in every particular individual being treated. Rather, a therapeutic dose may be a dose that produces a specific desired pharmacological response in a significant number of subjects when administered to patients requiring such treatment. In some embodiments, a reference to a therapeutic dose may refer to a dose measured in one or more specific tissues (e.g., tissues affected by the disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will understand that in some embodiments, a therapeutically effective amount of a particular agent or treatment may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses, for example, as part of a drug regimen.

[0115] Treatment: As used herein, the term “treatment” (also referred to as “to treat” or “to treat”) means the administration of a treatment administered for the purpose of partially or completely reducing, relieving, mitigating, inhibiting, delaying the onset of, reducing the severity of, and / or reducing the incidence of, or achieving any such result, one or more symptoms, characteristics, and / or causes of a particular disease, disorder, or condition. In some embodiments, such treatment may be for subjects who do not exhibit signs of the disease, disorder, or condition in question, and / or for subjects who exhibit only initial signs of the disease, disorder, or condition. Alternatively or additionally, such treatment may be for subjects who exhibit one or more established signs of the disease, disorder, and / or condition in question. In some embodiments, treatment may be for subjects who have been diagnosed with the disease, disorder, and / or condition in question. In some embodiments, treatment may be for subjects who have been found to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the disease, disorder, or condition in question.

[0116] II. Multispecific antigen-binding constructs This disclosure includes various binding agents comprising domains provided herein, specifically, binding agents comprising at least one APP-binding domain and at least one shielding domain that modulates APP binding by the APP-binding domain, wherein the shielding domain is linked to a proteolytically cleavable linker. In some embodiments, the binding agents encompassed by this disclosure may comprise at least one target cell-binding domain. In some embodiments, the binding agents encompassed by this disclosure may comprise two target cell-binding domains, for example, the two target cell-binding domains targeting the same epitope and / or antigen, or the two target cell-binding domains targeting different epitopes and / or antigens.

[0117] In some embodiments, any of the multispecific antigen-binding constructs may be therapeutic conjugates. The multispecific antigen-binding construct comprises multiple antigen-binding domains for binding to an antigen for therapeutic use. In embodiments herein, the multispecific construct comprises a first antigen-binding domain that binds to inhibit an antiphagocytic protein (APP), and a second antigen-binding domain that binds to the first antigen-binding domain to inhibit or reduce the interaction between the first antigen-binding domain and APP.

[0118] In some embodiments, the provided multispecific antigen-binding construct includes: (i) a first antigen-binding domain that binds to and inhibits an antiphagocytic protein (APP); (ii) a second antigen-binding domain that binds to the first antigen-binding domain and inhibits or reduces the interaction between the first antigen-binding domain and APP; (iii) a linker containing a proteolytically cleavable linker; (iv) a third antigen-binding domain that binds to a first target cell antigen; and (v) an immunoglobulin Fc region. In some embodiments, the linker containing a proteolytically cleavable linker links the second antigen-binding domain to the first antigen-binding domain or the immunoglobulin Fc region. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are linked by a linker containing a proteolytically cleavable linker. In some embodiments, the immunoglobulin Fc region and the second antigen-binding domain are linked by a linker containing a proteolytically cleavable linker.

[0119] In some embodiments, the binders encompassed by this disclosure include an antibody structure linked to an APP binding domain and a shielding domain. In some embodiments, the antibody structure may have the structure of any antibody or antibody fragment provided herein. In some embodiments, the antibody structure may have a quadruple-chain immunoglobulin structure or a variant thereof recognized in the art (e.g., those disclosed herein) comprising two immunoglobulin heavy chains and two immunoglobulin light chains, or essentially comprising them. In some embodiments, the antibody structure may include an immunoglobulin Fc domain. In some embodiments, the antibody structure may include an immunoglobulin Fc domain formed from the constant domains of two immunoglobulin heavy chains. In some embodiments, the Fc domain may be present in an antibody structure comprising two antibody-binding domains, each formed by the binding of a heavy chain variable domain and a light chain variable domain. In some embodiments, the Fc domain may be present in an antibody structure comprising two antibody-binding domains, each formed by the binding of a heavy chain variable domain present in an immunoglobulin heavy chain and a light chain variable domain present in an immunoglobulin light chain. In some embodiments, the Fc domain can be present in an antibody structure containing two antibody-binding domains, one or each of which are formed by the binding of a heavy-chain variable domain and a light-chain variable domain present in a fragment antigen-binding (Fab) domain. In some embodiments, the Fc domain can be present in an antibody structure containing two antibody-binding domains, one or each of which are formed by the binding of a heavy-chain variable domain and a light-chain variable domain present in an antibody fragment (such as scFv). In some embodiments, the Fc domain can be present in an antibody structure containing two antibody-binding domains, one or each of which are present in the variable domain of a heavy-chain-only antibody (VHH).

[0120] In some embodiments, the binders encompassed by this disclosure include an antibody structure in which an APP-binding domain is optionally linked via a linker at a first amino acid, and a shielding domain is optionally linked via a proteolytically cleavable linker at a second amino acid. In some embodiments, the binders encompassed by this disclosure include an antibody structure in which an APP-binding domain is optionally linked via a linker at a first amino acid, and the APP-binding domain is further linked via a proteolytically cleavable linker at its amino acid (e.g., the terminal amino acid of the APP-binding domain). It will be understood from this disclosure that the APP-binding domain and the shielding domain are arranged in the binder so that the shielding domain can interact (e.g., bind) with the APP-binding domain in a manner that reduces its APP-binding activity.

[0121] In some embodiments, when the linker containing a proteolytically cleavable linker is in an uncleaved state, the second antigen-binding domain inhibits or reduces the binding of the first antigen-binding domain to APP. In some embodiments, when the linker containing a proteolytically cleavable linker is proteolytically cleaved, the second antigen-binding domain does not interfere with the binding of the first antigen-binding domain to APP.

[0122] In some embodiments, the APP-binding domain is linked to the antibody structure of the binder disclosed herein via amino acids (e.g., terminal amino acids) of the Fc domain and / or heavy chain constant domain. In some embodiments, the APP-binding domain is linked to the antibody structure of the binder disclosed herein via amino acids (e.g., terminal amino acids) of the light chain constant domain. In some embodiments, the APP-binding domain is linked to the antibody structure of the binder disclosed herein via amino acids (e.g., terminal amino acids) of the light chain variable domain (e.g., a light chain variable domain present in Fab). In some embodiments, the APP-binding domain is linked to the antibody structure of the binder disclosed herein via a proteolytically cleavable linker.

[0123] In some embodiments, the target cell-binding domain is linked to the antibody structure of the conjugate disclosed herein via amino acids (e.g., terminal amino acids) of the Fc domain and / or heavy chain constant domain. In some embodiments, the target cell-binding domain is linked to the antibody structure of the conjugate disclosed herein via amino acids (e.g., terminal amino acids) of the light chain constant domain. In some embodiments, the target cell-binding domain is linked to the antibody structure of the conjugate disclosed herein via amino acids (e.g., terminal amino acids) of the light chain variable domain (e.g., a light chain variable domain present in Fab).

[0124] In some embodiments, the target cell-binding domain is linked to the antibody structure of the conjugate disclosed herein via a linker that does not contain motifs that are cleaved and / or substantially cleaved by human proteases.

[0125] In some embodiments, the target cell-binding domain is linked to the antibody structure of the conjugate disclosed herein via a linker that does not contain motifs that are cleaved and / or substantially cleaved by human proteases in the microenvironment under treatment.

[0126] In some embodiments, the target cell-binding domain is bound to the antibody structure of the conjugate disclosed herein via a linker that is cleaved by a protease known to cleave and / or substantially cleave a proteolytically cleavable linker bound to the APP-binding domain and / or does not contain a motif known to be substantially cleaved.

[0127] In some embodiments, the binders encompassed by this disclosure are labeled to facilitate detection. In some embodiments, labeling of the construct includes direct labeling of the construct by coupling (i.e., physically linking) it with a detectable substance, and indirect labeling of an antibody by reactivity with a detectable substance. Labeling and labeling methods are well known in the art and do not include, but are not limited to, radioactive agents, radioisotopes, fluorescent compounds, fluorophores (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)), chemiluminescent compounds, enzymes, enzyme cofactors, or any other labels known in the art. In some embodiments, enzymes that can be attached to the construct may include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase, and glucose oxidase (GOx). Fluorescent compounds may include, but are not limited to, ethidium bromide; fluorescein and its derivatives (e.g., FITC); cyanines and their derivatives (e.g., indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine); rhodamine; Oregon Green; eosin; Texas Red; Nile Red; Nile Blue; cresyl violet; oxazine 170; proflavin; acridine orange; acridine yellow; auramine; crystal violet; malachite green; porfin; phthalocyanine; bilirubin; allophycocyanin (APC); green fluorescent protein (GFP) and its variants (e.g., yellow fluorescent protein YFP, blue fluorescent protein BFP, and cyanide fluorescent protein CFP); ALEXIFLOUR® compounds (Thermo Fisher Scientific, Waltham, MA); and quantum dots. Other complexes that can be used as labels include biotin, avidin, and streptavidin.

[0128] A.APP binding domain The binding agents encompassed by this disclosure include domains that bind to antiphagocytic proteins (APP; APP-binding domains). The process of phagocytosis is naturally used to remove dying or pathogenic cells. Various mechanisms limit the frequency of phagocytosis in healthy cells, one of which is the balance between surface-expressed signaling molecules that promote or activate phagocytosis (sometimes referred to as "eat-me" signals) and other signaling molecules that suppress phagocytosis (sometimes referred to as "don't-eat-me" signals). On the other hand, when "don't-eat-me" signals that suppress phagocytosis are expressed by diseased cells, they can cause, contribute to, and / or exacerbate disease by enabling diseased cells to evade phagocytosis.

[0129] Eat-me signals include antibodies and complement opsonins, exposed phosphatidylserine (PS), calreticulin, oxidized low-density lipoproteins, cell-bound thrombospondin (TSP), modified intracellular adhesion molecules ICAM-3 and annexin I, and other modifications to surface proteins. Don't-eat-me signaling may include antiphagocytic receptors that mediate the recognition of don't-eat-me signals. For example, the CD47-SIRPα system is one of the most studied don't-eat-me checkpoints. In various cancers, cancer cells can overexpress CD47 and / or CD24, which enables immune evasion from macrophages.

[0130] Proteins involved in the "Don't Eat Me" signaling pathway can be referred to as APP. In some embodiments, APP is expressed on myeloid cells. In some embodiments, APP is expressed on cells targeted by phagocytosis (e.g., cancer cells). In some embodiments, APP is a protein other than the receptor or ligand of the "Don't Eat Me" signaling pathway that inhibits the phagocytic function of myeloid cells (e.g., LILRB2), and such APP may also be expressed on myeloid cells or on cells targeted by phagocytosis (e.g., cancer cells).

[0131] While we do not wish to be constrained by any particular scientific theory, CD47 expression on cells acts as a "self" marker that suppresses phagocytosis through its interaction with SIRPα. CD47 (also known as integrin-related protein, OV-3, and Rh-related protein) is a ubiquitously expressed, conserved 45-55 kDa transmembrane glycoprotein belonging to the Ig superfamily. CD47 has a single N-terminal extracellular immunoglobulin variable region (IgV) domain, followed by five hydrophobic transmembrane segments, and a short C-terminal cytoplasmic tail that undergoes alternative splicing to form four isoforms. SIRPα (also known as SIRPα1, PTPNS1, SHPS-1, BIT, p84, MFR, MyD-1, and CD172a) is a 115-120 kDa glycoprotein belonging to the SIRP paired receptor family. It is expressed in most tissues, for example, abundantly on monocytes, macrophages, CD8α classical type II dendritic cells (cDC2), neutrophils, and osteoclasts, as well as on microglia and neurons.

[0132] In addition to SIRPα and CD47, other receptors and ligands exist that can also act to suppress immune cell function (such as that of myeloid cells). For example, inhibitory receptors of the CD300 family, including CD31 (also known as PECAM-1) and CD300a and CD300f, can suppress phagocytosis. CD300a and CD300f are expressed on myeloid cells and bind to ligands such as phosphatidylserine (PS).

[0133] Siglec, or sialic acid-binding Ig-like lectins, is a large family of receptors that can inhibit phagocytosis, including, for example, CD33 (also called Siglec-3), CD22 (also called Siglec-2), and SIGLEC10. CD33 is expressed, for example, on myeloid cells and microglial cells. CD22 is expressed, for example, on B cells, as well as myeloid cells and microglia. SIGLEC10 is expressed on myeloid cells and some lymphocytes and is involved in signaling pathways along with CD24.

[0134] PD-1 (also known as programmed cell death protein 1 and CD279) is a well-known immunosuppressive receptor with two corresponding ligands known as PD-L1 (also known as CD274 and B7-H1, expressed, for example, on non-lymphoid cells) and PD-L2 (also known as CD273 and B7-DC, expressed, for example, on antigen-presenting cells). In addition to being expressed on lymphocytes, PD-1 expression can be induced in macrophages (e.g., by infection).

[0135] Human LILRB1 (leukocyte immunoglobulin-like receptor B1; also known as CD85J, ILT2, and LIR-1) is an inhibitory receptor expressed on myeloid and lymphoid cell subsets. LILRB1 binds to MHC class I molecules expressed on all nucleated cells, and the invariant β2-microglobulin (B2M) subunit of MHC class I has been shown to be important for this interaction. Specifically, without wishing to be constrained by any particular scientific theory, the β2-microglobulin (B2M) subunit of the MHC I complex mediates the interaction between MHC I and LILRB1. MHC class I expression can protect cells from phagocytosis via LILRB1 association.

[0136] In some embodiments, the APP binding domains contained herein bind to the APPs listed in the following table.

[0137] [Table 1] TIFF2026529669000003.tif218165TIFF2026529669000004.tif167165TIFF20265296690 00005.tif201165TIFF2026529669000006.tif228165TIFF2026529669000007.tif212165

[0138] In some embodiments, the APP binding domains encompassed by this disclosure inhibit or reduce the binding of APP (e.g., APP selected from programmed cell death 1 ligand 1 (PD-L1), programmed cell death 1 ligand 1 (PD-L2), CD47, CD24, β2-microglobulin (B2M), and major histocompatibility complex class I (MHC-I)) to its binding partner (e.g., a binding partner expressed by or present on myeloid cells (e.g., the binding partners disclosed herein)).

[0139] In some embodiments, the APP-binding domains encompassed by this disclosure inhibit or reduce the binding of APP (e.g., APP selected from PD-1, SIRPα, SIGLEC10, LILRB1, and LILRB2) to its binding partner (e.g., a binding partner expressed by or present on cells targeted for regulation (e.g., direct and / or indirect death) (e.g., cancer cells) (e.g., the binding partners disclosed herein)).

[0140] In some embodiments, the APP-binding domain encompassed by this disclosure binds to CD47 or SIRPα. In some embodiments, the APP-binding domain inhibits the interaction between CD47 expressed on cells targeted for regulation (e.g., direct and / or indirect death) and SIRPα expressed on myeloid cells (e.g., macrophages, dendritic cells, monocytes, or neutrophils). In some embodiments, the APP-binding domain encompassed by this disclosure binds to CD24 or SIGLEC10. In some embodiments, the APP-binding domain inhibits the interaction between CD24 expressed on cells targeted for regulation (e.g., direct and / or indirect death) and SIGLEC10 expressed on myeloid cells (e.g., macrophages, dendritic cells, monocytes, or neutrophils). In some embodiments, the APP-binding domain encompassed by this disclosure binds to a PD-1 ligand (such as PD-L1 and / or PD-L2) or PD-1. In some embodiments, the APP-binding domain inhibits the interaction between PD-1 ligand(s) expressed on cells targeted for regulation (e.g., direct and / or indirect death) and PD-1 expressed on myeloid cells (e.g., macrophages, dendritic cells, monocytes, or neutrophils). In some embodiments, the APP-binding domain encompassed by this disclosure binds to β2-microglobulin (B2M) or MHC-I or LILRB1. In some embodiments, the APP-binding domain inhibits the interaction between either B2M or MHC-I expressed on cells targeted for regulation (e.g., direct and / or indirect death) and LILRB1 expressed on myeloid cells (e.g., macrophages, dendritic cells, monocytes, or neutrophils). In other embodiments of the preceding example, LILRB2, rather than LILRB1, is the myeloid cell-expressed APP. In some embodiments, the APP-binding domain binds to LILRB2 expressed on myeloid cells (e.g., macrophages, dendritic cells, monocytes, neutrophils, tumor-associated macrophages (TAMs), tumor-infiltrating macrophages (TIMs), or myeloid-derived immunosuppressive cells (MDSCs)) to block the inhibition of phagocytosis by myeloid cells.

[0141] In some embodiments, the multispecific antigen-binding construct includes an antigen-binding domain. In some embodiments, the multispecific antigen-binding construct includes a first antigen-binding domain. In some embodiments, the first antigen-binding domain includes (i) the extracellular domain (ECD) of a cell surface-expressed protein, (ii) a binding fragment of the ECD of a cell surface-expressed protein, or (iii) a variant of the ECD or binding fragment of a cell surface-expressed protein that has been engineered to improve binding to APP. In some embodiments, the binding fragment of a cell surface-expressed protein includes a portion of the ECD of a cell surface-expressed protein. In some embodiments, the binding fragment of a cell surface-expressed protein includes the immunoglobulin-variable (V) region (domain 1) of the ECD of a cell surface-expressed protein. In some embodiments, the binding fragment of a cell surface-expressed protein is essentially derived from the immunoglobulin-variable (V) region (domain 1) of the ECD of a cell surface-expressed protein.

[0142] In some embodiments, a multispecific antigen-binding construct comprises a first antigen-binding domain comprising (i) a domain of wild-type SIRPα that binds to an antiphagocytic protein (APP), or (ii) a variant thereof comprising one or more amino acid substitutions in the domain of wild-type SIRPα that improves binding to APP, wherein the first antigen-binding domain is CD47, and a second antigen-binding domain which is an anti-SIRPα antibody or antigen-binding fragment that binds to the first antigen-binding domain and inhibits or reduces the interaction between the first antigen-binding domain and APP, wherein the first and second antigen-binding domains are linked by a proteolytically cleavable linker.

[0143] In some embodiments, the APP-binding domain binds to APP expressed by and / or bound to target cells that are the target of treatment. Binding agents encompassed by this disclosure, including the APP-binding domain, can induce phagocytosis of target cells.

[0144] In some embodiments, the binding agents encompassed by this disclosure include an APP-binding domain that binds to APP expressed by a cell or cell type that is associated with, characterized by, represents, causes, contributes to, and / or whose phagocytosis contributes to the treatment of a condition of interest. In some embodiments, the condition of interest is cancer. In some embodiments, cancer is selected from, but is not limited to, adenocarcinoma, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer (e.g., triple-negative or Her2-negative breast cancer), carcinoid cancer, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, glioma, head and neck cancer (e.g., head and neck squamous cell carcinoma), leukemia, liver cancer, lung cancer (e.g., NSCLC, SCLC), lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer (e.g., metastatic castration-resistant prostate cancer), kidney cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, and urothelial carcinoma.In some embodiments, cancers include, but are not limited to, cancers of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves, and other parts of the CNS (such as glioblastoma or medulloblastoma)); cancers of the head and / or neck, breast cancer, cancers of the circulatory system (e.g., cancers of the heart, mediastinum and pleura, as well as other intrathoracic organs, vascular cancers, and tumor-associated vascular tissue); cancers of the blood and lymphatic system (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt lymphoma, AIDS-associated lymphoma, malignant immunoproliferative disorders, multiple myeloma, and malignant plasmacytogenesis). Cancers of the body, lymphocytic leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell types, leukemias of unspecified cell types, unspecified malignant neoplasms of lymphoid tissue, hematopoietic tissue, and related tissues (such as diffuse large cell lymphoma, T-cell lymphoma, or cutaneous T-cell lymphoma); cancers of the excretory system (e.g., kidneys, renal pelvis, ureters, bladder, and other urinary tract); cancers of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectal tract, rectosigmoid junction, rectum, anus, and anal canal); cancers of the liver and intrahepatic bile ducts, gallbladder, and Cancers of other parts of the biliary tract, the pancreas, and other digestive organs; cancers of the oral cavity (e.g., lips, tongue, gums, floor of the mouth, palate, parotid glands, salivary glands, tonsils, oropharynx, nasopharynx, piriform sinus, hypopharynx, and other parts of the oral cavity); cancers of the reproductive system (e.g., vulva, vagina, cervix, uterus, ovaries, and other parts associated with the female reproductive organs, placenta, penis, prostate, testes, and other parts associated with the male reproductive organs); cancers of the airway (e.g., nasal cavity, middle ear, sinuses, larynx, trachea, bronchi, and lungs) (such as small cell lung cancer and non-small cell lung cancer); cancers of the skeletal system (e.g., Cancers of the bones and articular cartilage of the limbs, articular cartilage, and other sites; cancers of the skin (e.g., malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, mesothelioma, Kaposi's sarcoma); and cancers of other tissues including the peripheral and autonomic nervous systems, connective tissue and soft tissue, retroperitoneum and peritoneum, eyes and adnexa, thyroid gland, adrenal gland, and other endocrine glands and related structures; secondary and unspecified malignant neoplasms of lymph nodes; secondary malignant neoplasms of the respiratory and digestive systems; and secondary malignant neoplasms of other sites.

[0145] In some embodiments, the condition is selected from, for example, infection (e.g., bacterial and / or viral infection), atherosclerosis, cardiovascular disease (e.g., heart failure following myocardial infarction), autoimmune disease (e.g., systemic lupus erythematosus (SLE), autoimmune nephritis, autoimmune uveitis, or autoimmune heart valve disease), transplant organ rejection, fibrous disease, or neurological disease. In some embodiments, the binders encompassed by this disclosure include an APP-binding domain that binds to APP expressed by a cell or cell type further characterized by the expression of a target cell antigen encompassed by this disclosure.

[0146] In some embodiments, the APP-binding domain may be a receptor protein. In some embodiments, the APP-binding domain may be a fragment or domain of a receptor protein. In some embodiments, the APP-binding domain may be a receptor ligand. In some embodiments, the APP-binding domain may be a fragment or domain of a receptor ligand. In some embodiments, the APP-binding domain may be an antibody or antibody fragment. In some embodiments, the APP-binding domain may be a small molecule. In some embodiments, the APP-binding domain may be an aptamer.

[0147] In some embodiments, the APP-binding domain is the SIRPα protein. In some embodiments, the APP-binding domain is a fragment or domain of the SIRPα protein. In some embodiments, the APP-binding domain is the SIGLEC10 protein. In some embodiments, the APP-binding domain is a fragment or domain of the SIGLEC10 protein. In some embodiments, the APP-binding domain is the PD-1 protein. In some embodiments, the APP-binding domain is a fragment or domain of the PD-1 protein. In some embodiments, the APP-binding domain is the LILRB1 protein. In some embodiments, the APP-binding domain is a fragment or domain of the LILRB1 protein. In some embodiments, the APP-binding domain is the LILRB2 protein. In some embodiments, the APP-binding domain is a fragment or domain of the LILRB2 protein. In some embodiments, the APP-binding domain is the PD-L1 protein. In some embodiments, the APP-binding domain is a fragment or domain of the PD-L1 protein. In some embodiments, the APP-binding domain is the PD-L2 protein. In some embodiments, the APP-binding domain is a fragment or domain of the PD-L2 protein. In some embodiments, the APP-binding domain is the CD47 protein. In some embodiments, the APP-binding domain is a fragment or domain of the CD47 protein. In some embodiments, the APP-binding domain is the CD24 protein. In some embodiments, the APP-binding domain is a fragment or domain of the CD24 protein. In some embodiments, the APP-binding domain is the β2M protein. In some embodiments, the APP-binding domain is a fragment or domain of the β2M protein. In some embodiments, the APP-binding domain is a protein of the MHC-I complex (e.g., HLA-A, HLA-B, or HLA-C). In some embodiments, the APP-binding domain is a fragment or domain of a protein of the MHC-1 complex (e.g., HLA-A, HLA-B, or HLA-C).

[0148] In some embodiments, the APP-binding domain may include an antibody or its antigen-binding moiety. In some embodiments, the APP-binding antibody or its antigen-binding moiety may include at least one immunoglobulin heavy chain and / or at least one immunoglobulin light chain. In some embodiments, the APP-binding antibody or its antigen-binding moiety may include at least one immunoglobulin heavy chain variable domain and / or at least one immunoglobulin light chain variable domain. In some embodiments, the APP-binding antibody or its antigen-binding moiety may include at least one immunoglobulin heavy chain variable domain CDR1, CDR2, and CDR3, and / or at least one immunoglobulin light chain variable domain CDR1, CDR2, and CDR3.In some embodiments, the APP-conjugated antibody or its antigen-binding moiety is the extracellular domain (ECD) of a cell surface-expressed protein, a variant version of the ECD of a cell surface-expressed protein manipulated to improve binding to the target, an intrabody, a domain antibody, an antibody mime, Zybody®, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd' fragment, an Fd fragment, an isolated CDR or a set thereof, a single-chain antibody, a single-chain Fv (scFv), a disulfide-linked Fv (sdFv), a polypeptide-Fc fusion, a single-domain antibody (e.g., a shark single-domain antibody (IgNAR, etc.) or a fragment thereof), a camel antibody, a camelized antibody, a shielding antibody (e.g., Probody®), an afibody, an anti-idiotype (anti-Id) antibody This may include, or be, any antigen-binding fragment of any of the aforementioned, a body (e.g., an anti-anti-Id antibody), a single-stranded or tandem diabody (TandAb®), VHH, Anticalin®, Nanobody® minibody, BiTE®, ankyrin repeat protein or DARPIN®, Avimer®, DART, TCR-like antibody, Adnectin®, Affilin®, Trans-body®, Affibody®, TrimerX®, Microprotein, Fynomer®, Centyrin®, KALBITOR®, CAR, a modified TCR, or any of the aforementioned.

[0149] In some embodiments, the APP-binding domain is a variant APP-binding domain containing one or more amino acid substitutions that improve binding to CD47. In some embodiments, the binding to CD47 of a variant APP-binding domain containing one or more amino acid substitutions in the IgV domain is improved compared to the binding of a wild-type APP-IgV domain. In some embodiments, the APP-binding domain has a dissociation constant (K) less than 100 picomolar concentrations (pM). D) binds to CD47. In some examples, the variant APP binding domain dissociates at concentrations less than 100 picomoles (pM) with a dissociation constant (K D ) is used to combine with CD47.

[0150] In some embodiments, the affinity (K) of the APP-binding domain included by this disclosure to the target APP. D The binding affinity of the APP binding domain encompassed by this disclosure to the target APP may be about 0.002 to about 200 nM. In some embodiments, the binding affinity of the APP binding domain encompassed by this disclosure to the target APP may be about 250 nM, 200 nM, about 100 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 2 pM, or less. In some embodiments, the binding affinity is approximately 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 500 pM, 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, or 2 pM, or any range between these (e.g., approximately 5 nM to approximately 35 nM). In some embodiments, the binding affinity of the APP binding domains contained herein to the target APP is approximately 1 × 10⁻¹⁶ -7 Less than M (approximately 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 It may be less than M, or less than that, etc. In some embodiments, affinity (K D This can be determined using assays well known in the art, such as by using surface plasmon resonance (SPR) technology in BIACORE® assay instruments.

[0151] 1. CD47 APP binding domain Blocking the interaction between CD47 and endogenous SIRPα expressed on the surface of macrophages and dendritic cells can inhibit CD47 / SIRPα-mediated signaling, thereby eliminating CD47 / SIRPα-mediated inhibition of phagocytosis. Existing regulators of this pathway generally target ubiquitously expressed CD47 cell surface molecules (see, e.g., Chao et al. Cell 2010 Sep 3;142(5):699-713, Weiskopf et al. Science. 2013 Jul 5;341(6141):88-91). As described herein, CD47 APP-binding domains can be used to inhibit or block the interaction between CD47 and endogenous or wild-type SIRPα. For example, CD47 APP-binding domains that bind to CD47 can enable phagocytosis of solid tumor cells, inhibit tumor growth, and block metastatic tumor cells. This specification provides multispecific antigen-binding constructs containing a CD47 APP-binding domain. Such multispecific antigen-binding constructs have specificity for CD47 via the CD47-binding domain and may also include binding domains having specificity for other antigens. In some embodiments, the multispecific antigen-binding construct contains a CD47 APP-binding domain comprising a SIRPα polypeptide or a variant or fragment thereof. In other embodiments, the multispecific antigen-binding construct contains a CD47 APP-binding domain that is not a SIRPα polypeptide. In these embodiments, the multispecific antigen-binding construct may include a CD47 APP-binding domain that is an anti-CD47 antibody or an antigen-binding fragment thereof. In embodiments of the multispecific antigen-binding constructs provided herein, the first antigen-binding domain is a CD47-binding domain (such as SIRPα).

[0152] (i)SIRPα SIRPα protein is a membrane glycoprotein expressed by neurons and myeloid cells and is abundantly present on macrophages. The full-length SIRPα protein contains a signal peptide, an extracellular domain (ECD), and a cytoplasmic region. The SIRPα extracellular domain (ECD) contains a single N-terminal IgV-like domain (IgV domain), followed by two IgC-like domains, a transmembrane domain, and a cytoplasmic tail. The IgV domain (D1) of SIRPα can interact with CD47. The interaction between the N-terminal IgV domain of CD47 and SIRPα IgV / D1 promotes phosphorylation of tyrosine residues. An exemplary full-length SIRPα protein is shown in Uniprot accession number P78324.

[0153] CD47 functions as a ligand for SIRPα. When endogenous SIRPα on the surface of macrophage cells binds to CD47, SIRPα elicits a signal that inhibits phagocytosis of CD47-containing cells. The binding interface between SIRPα and CD47, and the residues of both proteins involved in binding, are known and have been described in the art (see, for example, Hatherley et al. (2007) J. Biol. Chem. 282:14567-75, Nakaishi et al. (2008) J. Mol. Biol. 375:650-60). As shown herein, the binding of a multispecific antigen-binding construct containing a SIRPα polypeptide, such as a variant SIRPα polypeptide (e.g., a variant SIRPα polypeptide with increased affinity for CD47 compared to wild-type SIRPα), can be used to disrupt the binding of endogenous SIRPα to CD47 on the cell surface. SIRPα polypeptides (such as those provided herein) can bind to CD47 and inhibit the interaction between CD47 and endogenous or wild-type SIRPα.

[0154] In some embodiments, the multispecific antigen-binding construct includes an antigen-binding protein (such as SIRPα) that binds to the antiphagocytic protein (APP), which is CD47. In the embodiments herein, the antigen-binding protein is SIRPα that binds to the antiphagocytic protein (APP), which is CD47. In some embodiments, the SIRPα polypeptide binds to CD47 and inhibits the interaction between CD47 and SIRPα. In some embodiments, SIRPα binds to CD47 on diseased cells (e.g., tumor cells) with higher affinity compared to CD47 on non-disease cells.

[0155] In some embodiments, SIRPα in the multispecific antigen-binding constructs provided herein inhibits the binding of the extracellular domain or binding fragment of the wild-type SIRPα polypeptide to CD47. In some embodiments, SIRPα provided herein inhibits the binding of wild-type SIRPα to the IgSF domain of the CD47 protein. In the embodiments provided herein, CD47 is the human CD47 protein.

[0156] In several embodiments, the SIRPα in the multispecific antigen-binding construct comprises a SIRPα polypeptide selected from (i) the ECD of wild-type SIRPα, (ii) a binding fragment of wild-type SIRPα, and (iii) a variant of the ECD or binding fragment of wild-type SIRPα that has been modified to improve binding to CD47.

[0157] In some embodiments, the multispecific antigen-binding construct comprises the wild-type SIRPα polypeptide or a portion thereof. In some embodiments, SIRPα in the multispecific antigen-binding construct comprises the domain of wild-type SIRPα. In some embodiments, SIRPα in the multispecific antigen-binding construct comprises the extracellular domain (ECD) of wild-type SIRPα. In some embodiments, the SIRPα polypeptide is the wild-type SIRPα polypeptide essentially derived from the extracellular domain of SIRPα. In some embodiments, SIRPα is a binding fragment of wild-type SIRPα that binds to CD47. In some embodiments, SIRPα is a binding fragment of wild-type SIRPα ECD that binds to CD47. In some embodiments, the binding fragment of SIRPα comprises the immunoglobulin variable (V) region (also called IgV; D1) of the ECD of SIRPα. In some embodiments, the SIRPα polypeptide is a binding fragment of wild-type SIRPα polypeptide that comprises the SIRPα IgV domain and binds to CD47. In any of the prior embodiments, the SIRPα polypeptide is the APP-binding domain.

[0158] In some embodiments, the multispecific antigen-binding construct comprises a variant SIRPα polypeptide. The variant SIRPα polypeptide may comprise one or more amino acid modifications in the unmodified SIRPα polypeptide (such as any wild-type SIRPα polypeptide provided herein). For example, the variant SIRPα polypeptide provided herein comprises one or more amino acid substitutions (alternatively, “mutations” or “replacements”), deletions, or additions in the unmodified SIRPα polypeptide (such as wild-type SIRPα polypeptides containing an extracellular domain, such as the wild-type SIRPα polypeptide described herein). One or more amino acid modifications (e.g., substitutions) may be located in the extracellular domain of the reference (e.g., unmodified or wild-type) SIRPα sequence. In some embodiments, one or more amino acid modifications are located in D1 of SIRPα.

[0159] Unless otherwise stated, as shown throughout this disclosure, amino acid modifications (or multiple modifications) in the variant SIRPα polypeptide are designated by amino acid position numbers corresponding to the sequence of the unmodified or wild-type SIRPα ECD shown in SEQ ID NO: 206 or the position numbering of a portion thereof shown in SEQ ID NO: 103 or 104. Identifying the corresponding positions of modifications (e.g., amino acid substitutions) in the SIRPα polypeptide (including a portion thereof containing its IgV domain) is within the scope of the art, such as by alignment of the variant SIRPα sequence with the amino acid sequence shown in SEQ ID NO: 103 or 104. With respect to amino acid substitutions throughout this disclosure, the amino acid position is shown in the center, the corresponding reference (e.g., unmodified or wild-type) amino acid is listed before the number, and the variant amino acid substitution is listed after the number. For example, the amino acid substitution N80A refers to the substitution of asparagine (N) at position 80 of alanine (A), where position 80 is the 80th amino acid in the amino acid sequence shown in SEQ ID NO: 103 or 104.

[0160] In some embodiments, the SIRPα into which the amino acid modification is introduced is wild-type SIRPα (such as any wild-type SIRPα described herein) containing the extracellular domain of SIRPα. In exemplary embodiments, variant SIRPα comprises one or more amino acid modifications in wild-type SIRPα containing the extracellular domain of SIRPα. On the other hand, the variant SIRPα polypeptide does not need to contain the entire extracellular domain (ECD). In some embodiments, variant SIRPα comprises one or more amino acid modifications in wild-type SIRPα containing a portion of the ECD of SIRPα (such as a binding fragment of SIRPα that binds to CD47). In some embodiments, one or more amino acid modifications (e.g., substitutions) are present in the SIRPα ECD or in a portion thereof containing IgV. In some embodiments, variant SIRPα comprises one or more amino acid modifications in wild-type SIRPα containing the IgV domain of SIRPα. In some embodiments, variant SIRPα comprises one or more amino acid modifications in a binding fragment of the SIRPα ECD containing D1. In some embodiments, variant SIRPα includes one or more amino acid modifications in the amino acid sequence shown in SEQ ID NO: 103 or 104. In some embodiments, variant SIRPα includes one or more amino acid modifications in the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, variant SIRPα includes one or more amino acid modifications in the amino acid sequence shown in SEQ ID NO: 103.

[0161] In some embodiments, the variant SIRPα polypeptide is a soluble polypeptide and lacks a transmembrane domain.

[0162] In embodiments of this specification, the variant SIRPα polypeptide(s) exhibit a change (e.g., an increase) in binding affinity to CD47. In some embodiments, the variant SIRPα binds to CD47 with higher affinity than wild-type SIRPα. In embodiments of this specification, the variant SIRPα includes one or more amino acid substitutions in wild-type SIRPα that alter binding to CD47. In some embodiments, the variant SIRPα includes one or more amino acid substitutions in the domain of wild-type SIRPα that improves binding to CD47. In certain embodiments, the variant SIRPα includes one or more amino acid substitutions in the extracellular domain of wild-type SIRPα or in the binding fragment of the SIRPα ECD, where the one or more amino acid substitutions improve binding to CD47 compared to the binding of wild-type SIRPα that does not contain the one or more amino acid substitutions. In embodiments of this specification, the variant SIRPα includes a binding fragment of wild-type SIRPα that has been manipulated to improve binding to CD47. In some embodiments, the binding fragment of SIRPα includes D1. In the embodiments described herein, variant SIRPα comprises the extracellular domain of wild-type SIRPα that has been modified to improve its binding to CD47. In the embodiments described herein, variant SIRPα essentially consists of the extracellular domain of wild-type SIRPα that has been modified to improve its binding to CD47. The improved binding of variant SIRPα compared to wild-type or unmodified SIRPα can be demonstrated by the higher affinity that variant SIRPα exhibits for CD47 compared to wild-type SIRPα. The increased affinity of variant SIRPα is reflected in the higher dissociation constant (K) compared to wild-type SIRPα. D This can be demonstrated by the small size of the dissociation constant. Therefore, the smaller the dissociation constant, the tighter the binding to the ligand (e.g., CD47) or the higher the affinity between the ligand and the protein (e.g., between CD47 and SIRPα). In some embodiments, variant SIRPα binds to CD47 on diseased cells (e.g., tumor cells) with higher affinity compared to CD47 on non-disease cells.

[0163] In some embodiments, SIRPα provided herein (e.g., variant SIRPα) binds to CD47, blocking the binding of wild-type or endogenous SIRPα to CD47. For example, variant SIRPα and wild-type SIRPα (e.g., endogenous) polypeptides may "compete" for the same CD47 epitope. In some examples, variant SIRPα or wild-type SIRPα in a multispecific antigen-binding construct may be superior to wild-type or endogenous SIRPα in binding to CD47. In some embodiments where variant SIRPα is superior to wild-type (e.g., endogenous) SIRPα in binding to CD47, variant SIRPα has increased affinity for CD47 compared to wild-type (e.g., endogenous) SIRPα.

[0164] In embodiments where the SIRPα polypeptide includes a SIRPα binding fragment, the fragment or portion of the SIRPα polypeptide is sufficient to bind to CD47. In some of the embodiments, the specific binding fragment is less than the full-length ECD shown in SEQ ID NO: 206. In some embodiments, the SIRPα binding fragment (also referred herein to as the SIRPα binding region) includes the immunoglobulin variable (V) region (domain 1) of the ECD of SIRPα. In some embodiments, the SIRPα binding fragment contains the sequence shown in SEQ ID NO: 103. In some embodiments, the SIRPα binding fragment contains the sequence shown in SEQ ID NO: 104. In some embodiments, the SIRPα binding fragment is essentially derived from the immunoglobulin variable (V) region (domain 1) of the ECD of SIRPα. In some embodiments, the SIRPα binding fragment is essentially derived from the sequence shown in SEQ ID NO: 103. In some embodiments, the SIRPα binding fragment contains, or is essentially derived from, the sequence shown in SEQ ID NO: 104. In some cases, the IgV domain is the SIRPα binding fragment. In some embodiments, the SIRPα binding fragment is or contains amino acids 1 to 115 of SEQ ID NO: 206.

[0165] In some embodiments, SIRPα is a SIRPα-binding region with an amino acid length of 100-120 (e.g., 105-115, 105-110, 105-115, 110-120, 110-115, 115-120). In some embodiments, the SIRPα-binding region has an amino acid length of 106-118. In some embodiments, the SIRPα-binding region has an amino acid length of 112-118. In embodiments of this specification, the SIRPα-binding region has an amino acid length of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120, or about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. In some examples, the SIRPα-binding region has an amino acid length of 115 or about 115.

[0166] In some embodiments, wild-type SIRPα is wild-type human SIRPα. In some embodiments, variant SIRPα comprises one or more amino acid modifications (e.g., one or more amino acid substitutions) in wild-type human SIRPα. In embodiments herein, wild-type human SIRPα is any allele of human SIRPα (see, for example, Takenaka Nat Immunol. 2007; 8(12): 1313-1323; GenBank accession numbers NM001040022.1 and D86043.1). In embodiments herein, the IgV domain of wild-type human SIRPα is as shown in SEQ ID NOs. 103 and 104. In embodiments herein, the ECD of wild-type human SIRPα is as shown in SEQ ID NOs. 206.

[0167] In some embodiments, the variant SIRPα polypeptide has up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty amino acid modifications (e.g., substitutions) in the wild-type SIRPα sequence. In some embodiments, the variant SIRPα polypeptide has up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty amino acid substitutions in the ECD or its specific binding fragment of the wild-type TACI sequence. The modifications (e.g., substitutions) may be present in the IgV domain. In some embodiments, the variant SIRPα polypeptide has up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty amino acid substitutions in the IgV domain of the wild-type SIRPα sequence.

[0168] In some embodiments, SIRPα comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the wild-type SIRPα polypeptide, wherein the wild-type SIRPα polypeptide comprises (i) the amino acid sequence shown in SEQ ID NO: 206 or (ii) a portion of SIRPα ECD containing the IgV domain of SIRPα. In certain embodiments, SIRPα comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the wild-type SIRPα polypeptide, wherein the wild-type SIRPα polypeptide comprises the IgV domain of SIRPα having the sequence of amino acids 1-112, 1-113, 1-114, 1-115, 1-116, 1-117, 1-118, 1-119, or 1-120 of SEQ ID NO: 206.

[0169] In some embodiments, SIRPα comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 104.

[0170] In some embodiments, SIRPα comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103.

[0171] In some embodiments, variant SIRPα comprising one or more described amino acid modifications (e.g., amino acid substitutions) has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the SIRPα polypeptide or its specific binding fragment shown in SEQ ID NO: 103 or its domain.

[0172] In some embodiments, variant SIRPα comprising one or more described amino acid modifications (e.g., amino acid substitutions) has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the SIRPα polypeptide or its specific binding fragment shown in SEQ ID NO: 104.

[0173] In some embodiments, SIRPα is a variant SIRPα that includes one or more amino acid substitutions in the IgV domain of wild-type SIRPα, which improves binding to CD47. In some embodiments, the binding of the variant SIRPα with one or more amino acid substitutions in the IgV domain to CD47 is improved compared to the binding of the wild-type SIRPα IgV domain.

[0174] In some embodiments, the SIRPα-binding domain has a dissociation constant (K) of less than 100 nanomolar (nM), less than 10 nM, less than 1 nM, less than 100 picomolar (pM), less than 10 pM, or less than 1 pM, or any combination of the aforementioned. D It binds to CD47 via ). Specific SIRPα-binding domains can be selected based on the desired binding affinity. This provides a platform that allows for flexible selection of interactions, such as depending on the specific shielding domain to be used. In some embodiments, the binding affinity of the SIRPα-binding domain (such as a variant SIRPα-binding domain) to CD47 is approximately 250 nM, 200 nM, 100 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 500 pM, 100 pM, 60 pM, 50 pM, 20 pM, 15 pM, 10 pM, 5 pM, 2 pM, or less. In some embodiments, the binding affinity is approximately 250 nM, approximately 200 nM, approximately 100 nM, approximately 50 nM, approximately 30 nM, approximately 20 nM, approximately 10 nM, approximately 7.5 nM, approximately 7 nM, approximately 6.5 nM, approximately 6 nM, approximately 5 nM, approximately 4.5 nM, approximately 4 nM, approximately 3.5 nM, approximately 3 nM, approximately 2.5 nM, approximately 2 nM, approximately 1.5 nM, approximately 1 nM, approximately 500 pM, approximately 100 pM, approximately 50 pM, approximately 20 pM, approximately 10 pM, approximately 5 pM, or approximately 2 pM, or any range between these (e.g., approximately 5 nM to approximately 35 nM). In some embodiments, the binding affinity of the CD47 variant SIRPα is 1 × 10⁻¹⁶ -7 M or approximately 1 x 10 -7 Less than M (approximately 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 It is less than M, or less than that, etc. In some embodiments, affinity (K DThis can be determined using assays well known in the art, such as by using surface plasmon resonance (SPR) technology in BIACORE® assay instruments.

[0175] For example, in some embodiments, the SIRPα-binding domain is either a wild-type SIRPα-binding domain or a wild-type human CD47 (such as cell surface-expressed CD47) with a dissociation constant (K) of less than 100 nanomolar (nM). D This is the variant that is joined by ). In some embodiments, K D These ranges are 1nM~100nM, 1nM~75nM, 1nM~50nM, 1nM~25nM, 1nM~10nM, 10nM~100nM, 10nM~75nM, 10nM~50nM, 10nM~25nM, 25nM~100nM, 25nM~75nM, 25nM~50nM, or 50nM~100nM, 50nM~75nM, or 75nM~100nM.

[0176] In some embodiments, the SIRPα-binding domain is a variant SIRPα containing one or more amino acid substitutions in the domain of wild-type SIRPα (e.g., the IgV domain) that improves binding to CD47. In some embodiments, the variant SIRPα has a moderate binding affinity to CD47. In some embodiments, the variant SIRPα has a dissociation constant (K) less than 1 nM. D ) binds to wild-type human CD47 (such as cell surface-expressed SIRPα). In some embodiments, K D These ranges are 100pM~1nM, 100pM~750pM, 100pM~500pM, 100pM~250pM, 250pM~1nM, 250pM~750pM, 250pM~500pM, 500pM~1nM, 500pM~750pM, or 750pM~1nM. An example of such a variant SIRPα is SIRPα containing the mutation E54Q, which in some cases contains this mutation as the sole mutation compared to the domain of wild-type SIRPα.

[0177] In some embodiments, the SIRPα-binding domain is a variant SIRPα containing one or more amino acid substitutions in the wild-type SIRPα domain (e.g., the IgV domain) that improves binding to CD47. In some embodiments, the variant SIRPα has a high or relatively high affinity for CD47. In some embodiments, the variant SIRPα has a dissociation constant (K) less than 100 picomolar concentrations (pM). D ) binds to wild-type human CD47 (such as cell surface-expressed SIRPα). In some embodiments, K D The range is 1 pM to 100 pM, and optionally 1 pM to 75 pM, 1 pM to 50 pM, 1 pM to 25 pM, 1 pM to 10 pM, 10 pM to 100 pM, 10 pM to 75 pM, 10 pM to 50 pM, 10 pM to 25 pM, 25 pM to 100 pM, 25 pM to 75 pM, 25 pM to 50 pM, or 50 pM to 100 pM, 50 pM to 75 pM, or 75 pM to 100 pM. An example of such a variant is the variant SIRPα, known as CV1. In some embodiments, variant SIRPα has the amino acid substitutions V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), and V92I (corresponding to the amino acid numbering in SEQ ID NO: 103 or SEQ ID NO: 104).

[0178] In some embodiments, the SIRPα-binding domain (e.g., variant SIRPα-binding domain) has a dissociation constant (K) of 1 pM to 100 pM or approximately 1 pM to 100 pM, optionally 10 pM to 50 pM or approximately 10 pM to 50 pM. D ) is used to combine with CD47.

[0179] The variant SIRPα polypeptides provided herein may include one or more amino acid modifications known in the art to alter (e.g., improve) binding to CD47. Exemplary amino acid modifications (e.g., substitutions) are described in Lee et al., J Immunol. 2007 Dec 1;179(11):7741-50, Weiskopf et al., Science. 2013 Jul 5;341(6141):10.1126 / science.1238856, and International Patent Publication No. WO2016 / 023040. In some embodiments, the variant SIRPα includes amino acid substitutions at the contact residue with CD47. In several embodiments, variant SIRPα includes amino acid substitutions at one or more contact residues at positions A29, L30, I31, P32, V33, G34, P35, Q52, K53, E54, S66, T67, K68, R69, F74, K93, K96, G97, S98, and D100. In some embodiments, variant SIRPα includes amino acid substitutions at the hydrophobic core. In several embodiments, variant SIRPα includes amino acid substitutions at one or more of the hydrophobic cores at positions L4, V6, V27, I36, F39, L48, I49, Y50, F57, V60, M72, F74, I76, V92, F94, and F103.In some embodiments, variant SIRPα is V27I or V27L, K53R, S66T or S66G, K68R, and F103V;L4V or L4I, V27I or V27L, E47V or E47L, K53R, E54Q, S66T or S66G, K68R, V92I, and F103V;L4V or L4I, V6I or V6L, A21V, V27I or V27L, I31T, I31S or I31F, E47V or E47L, K53R, H56P or H56R, S66 T or S66G, K68R, and F94L or F94V; V6I or V6L, V27I or V27L, I31T, I31S, or I31F, E47V or E47L, K53R, E54Q, H56P or H56R, S66T or S66G, V92I, and F94L or F94V; L4V or L4I, A21V, V27I or V27L, I31T, I31S, or I31F, E47V or E47L, K53R, E54Q, H56P or H56R, S66T or S66G, F94 L or F94V, and F103V; L4V or L4I, V6I or V6L, V27I or V27L, I31T, I31S, or I31F, E47V or E47L, K53R, H56P or H56R, S66T or S66G, K68R, V92I, and F94L or F94V; L4V or L4I, V6I or V6L, I31T, I31S, or I31F, E47V, or E47L, K53R, H56P or H56R; S66T, or S66G, V92I, and F103V; V Includes combinations of amino acid substitutions selected from 6I, V27I, I31F, E47L, K53R, E54Q, H56P, and S66T;L4V, V6I, V27I, 131F, E47V, K53R, E54Q, H56P, V63I, S66T, K68R, and V92I;V6I, V27I, I31T, E47V, K53R, E54Q, H56P, S66G, K68R, V92I, and F103V;V6I, V27I, 131F, E47V, K53R, E54Q, H56P, S66T, and V92I.

[0180] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in the wild-type SIRPα polypeptide or its specific binding fragment, selected from L4F or L4I or L4V, V6F or V6I or V6L, V27F or V27I or V27L, I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L or H56R, S66G or S66T or S66A, K68R, N80A, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V, or their conserved amino acid substitutions (corresponding to the amino acid numbering of SEQ ID NO: 103). In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in the wild-type SIRPα polypeptide or specific binding fragment selected from L4F or L4I or L4V, V6F or V6I or V6L, A27F or A27I or A27L, I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L or H56R, L66G or L66T or L66A, K68R, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V, or their conserved amino acid substitutions (corresponding to the amino acid numbering of SEQ ID NO: 104).

[0181] A conservative amino acid modification (e.g., substitution) is any amino acid that belongs to the same amino acid class as the amino acid being substituted, other than the reference (e.g., unmodified) or wild-type amino acid. The classes of amino acids are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartic acid, glutamic acid, asparagine, and glutamine).

[0182] In some embodiments, the variant SIRPα polypeptide has one or more amino acid substitutions in the wild-type SIRPα polypeptide or the specific binding fragment, where the one or more amino acid substitutions are K53R, E54Q, and S66T (corresponding to the amino acid numbering in SEQ ID NO: 103).

[0183] In some embodiments, the variant SIRPα polypeptide has one or more amino acid substitutions in the wild-type SIRPα polypeptide or the specific binding fragment, where the one or more amino acid substitutions are K53R, E54Q, and L66T (corresponding to the amino acid numbering in SEQ ID NO: 104).

[0184] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in the wild-type SIRPα polypeptide or specific conjugate, where one or more substitutions are V6I, V27I, I31F, E47V, K53R, E54Q, H56P, S66T, and V92I; or V6I, V27I, I31F, E47L, K53R, E54Q, H 56P and S66T; or L4V, V6I, V27I, I31F, E47V, K53R, E54Q, H56P, V63I, S66T, K68R, and V92I; or V6I, V27I, I31T, E47V, K53R, E54Q, H56P, S66G, K68R, V92I, and F103V (corresponding to the amino acid numbering of SEQ ID NO: 103).

[0185] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in the wild-type SIRPα polypeptide or specific conjugate, where one or more substitutions are V6I, A27I, I31F, E47V, K53R, E54Q, H56P, L66T, and V92I; or V6I, A27I, I31F, E47L, K53R, E54Q, H 56P and L66T; or L4V, V6I, A27I, I31F, E47V, K53R, E54Q, H56P, V63I, L66T, K68R, and V92I; or V6I, A27I, I31T, E47V, K53R, E54Q, H56P, L66G, K68R, V92I, and F103V (corresponding to the amino acid numbering of SEQ ID NO: 104).

[0186] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in the wild-type SIRPα polypeptide or a specific conjugate, where one or more substitutions are V6I, V27I, I31F, E47V, K53R, E54Q, H56P, S66T, and V92I (corresponding to the amino acid numbering in SEQ ID NO: 103).

[0187] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in the wild-type SIRPα polypeptide or a specific conjugate, where one or more substitutions are V6I, A27I, I31F, E47V, K53R, E54Q, H56P, L66T, and V92I (corresponding to the amino acid numbering in SEQ ID NO: 104).

[0188] In some embodiments, the variant SIRPα polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105.

[0189] In some embodiments, the variant SIRPα polypeptide is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105.

[0190] In some embodiments, the variant SIRPα polypeptide comprises the amino acid sequence shown in SEQ ID NO: 105.

[0191] In some embodiments, the variant SIRPα polypeptide is represented by the amino acid sequence shown in SEQ ID NO: 105.

[0192] In some embodiments, the SIRPα polypeptide is deglycosylated.

[0193] In some embodiments, SIRPα contains an N-glycosylation site N80 in the D1 region that has been mutated to alanine (A). In some embodiments, the N-glycosylation site N80 is not mutated, and the glycosylation site is retained.

[0194] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in the wild-type SIRPα polypeptide or a specific conjugate, where one or more substitutions include N80A (corresponding to the amino acid numbering in SEQ ID NO: 103 or SEQ ID NO: 104).

[0195] In some embodiments, the variant SIRPα polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10.

[0196] In some embodiments, the variant SIRPα polypeptide is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10.

[0197] In some embodiments, the variant SIRPα polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27.

[0198] In some embodiments, the variant SIRPα polypeptide is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27.

[0199] In some embodiments, the variant SIRPα polypeptide comprises the amino acid sequence shown in SEQ ID NO: 27.

[0200] In some embodiments, the variant SIRPα polypeptide is represented by the amino acid sequence shown in SEQ ID NO: 27.

[0201] (ii) Anti-CD47 antibody It has been previously shown that blocking CD47 activity with an anti-CD47 antibody activates macrophage phagocytosis of CD47+ cells (such as tumor cells). Administration of an anti-CD47 monoclonal antibody can reduce tumor burden in mouse models of hematological neoplasms and solid tumors. In embodiments herein, a multispecific antigen-binding construct may contain an anti-CD47 antibody or its antigen-binding fragment. For example, an anti-CD47 antibody developed to date and / or known in the art may be included in the multispecific antigen-binding construct provided herein. In some embodiments, the anti-CD47 antibody of this disclosure blocks the binding between the extracellular domain of a SIRP-α polypeptide (e.g., ECD including the D1 domain) and the IgSF domain of a human CD47 polypeptide. For example, the anti-CD47 antibody and the SIRP-α polypeptide may "compete" for the same CD47 epitope, and / or the binding of the antibody to CD47 may be mutually exclusive with the binding of CD47 to SIRP-α.

[0202] One of the numerous publicly available and / or known anti-CD47 agents may be included in the constructs described herein. Exemplary anti-CD47 antibodies or their antigen-binding fragments include, but are not limited to, Hu5F9-G4 (also known as maglorimab and 5F9 (by Gilead Sciences, Inc.)), a humanized IgF4 monoclonal antibody with high affinity for CD47 (see Liu J, Wang L, Zhao F, et al. PLoS One 10:e0137345 (2015)); CC-90002 (Celegene; Clinical Trials.gov identifiers NCT02641002 and NCT02367196), a humanized IgF4 anti-CD47 monoclonal antibody (see Narla Abstract 4694, Immunology. 2017 4694-4694); and AO-176 (Arch Oncology; Clinical Trials.gov identifier) ​​a humanized IgF2 anti-CD37 mAb. See NCT04445701; Puro.Mol.Cancer Ther.2020;19(3):835-846) and related Vx1000R mouse anti-human CD47 antibody (see Kaur.Antibody Ther.2020;3(3):179-192); and the human IgG4 anti-CD47 mAb SRF231 (manufactured by Surface Oncology).See ClinicalTrials.gov identifier NCT03512340; Holland.Blood.2016;128(22)1843-1843); IMC-002 (manufactured by ImmuneOncia Therapeutics; ClinicalTrials.gov identifier NCT04306224; Yoo, J.Immunother.Cancer.2020;8(Suppl 3)A237-A237), a fully human IgG4 anti-CD47 mAb; Letaplimab (manufactured by Innovent Biologics; see ClinicalTrials.gov identifier NCT0376149); SHR-1603 (by Jiangsu Hengrui Medicine, Co., Ltd.); TJC4 (manufactured by I-Mab Biopharma, Co., Ltd.); IBI188 (Innovent This includes Biologics, Inc.'s AO-176 (Arch Oncology, Inc.) or any variant or combination thereof. Other examples include anti-CD47 monoclonal antibodies previously shown to block the CD47-SIRP interaction, which may be included in the multispecific antigen-binding constructs provided herein, such as B6H12.2 and BRIC126 (see, e.g., Subramanian et al., Blood. 2006;107:2548-2556). Other examples include the multispecific antigen-binding constructs provided herein, which may contain Hu5F9-G4, a humanized IgG4 monoclonal antibody shown to block the CD47-SIRPα interaction (ClinicalTrials.gov identifier NCT02953509). Anti-CD47 antibody or antibody fragment.

[0203] In some embodiments, the multispecific antigen-binding constructs provided herein contain anti-CD47 single-domain antibody fragments (such as those derived from camel heavy chain antibodies (nanobodies, VHH domains)). In several embodiments, previously developed VHH domains can be included in the multispecific antigen-binding constructs provided herein. For example, HuNb1, which is a VHH having high affinity for CD47.

[0204] In embodiments of this specification, the multispecific antigen-binding construct may contain a panreactive anti-CD47 antibody or its antigen-binding fragment. For example, the multispecific antigen-binding construct provided herein contains an anti-CD47 antibody or its antigen-binding fragment that has affinity for wild-type CD47 and also affinity for other CD47 variants.

[0205] B. Shielding Domain The binding agents encompassed by this disclosure may include a shielding domain that modulates the binding of APP by an APP-binding domain. In some embodiments, the shielding domain, upon binding, binds to the APP-binding domain in a manner that inhibits its binding to APP. The disclosure further includes the possibility of binding the shielding domain to a proteolytically cleavable linker, so that the shielding domain inhibits APP binding when bound to the binding agent (i.e., when the linker is not cleaved), but the inhibition of APP binding is removed when the linker is cleaved, thereby achieving activatable activity. Thus, the binding agents encompassed by this disclosure include an APP-binding domain and a shielding domain that binds to the APP-binding domain, and the modulation of myeloid cell activity may be conditional on the cleavage of a proteolytically cleavable linker. Furthermore, if one or more proteases capable of cleaving a proteolytically cleavable linker are more active or more highly expressed in a particular microenvironment (e.g., a disease microenvironment such as a tumor microenvironment), the activity of the binding agent may be specific to a particular microenvironment.

[0206] In embodiments of the multispecific antigen-binding constructs provided herein, the second antigen-binding domain is a shielding domain that modulates the binding of APP by the APP-binding domain. In some embodiments of the multispecific antigen-binding constructs provided herein, the second antigen-binding domain is a shielding domain that modulates the binding of CD47 to SIRPα.

[0207] In some embodiments, a first exemplary state of the binder may be one in which the proteolytically cleavable linker is intact, and as a result, the bound shielding domain can inhibit APP binding by the APP binding domain. In some embodiments, a second exemplary state of the binder may be one in which the proteolytically cleavable linker is cleaved, and the shielding domain is released from the binder. In some embodiments, the first exemplary state is characterized in that the binder does not induce or significantly induces myeloid cell activity (e.g., phagocytosis of target cells) against target cells. In some embodiments, the second exemplary state is characterized in that the binder can induce myeloid cell activity (e.g., phagocytosis of target cells) against target cells.

[0208] In some embodiments, the first and second exemplary states demonstrate that the effects on myeloid cells and / or target cells (e.g., direct and / or indirect target cell death, e.g., target cell phagocytosis) differ between activation conditions (e.g., an activated microenvironment) and deactivation conditions (e.g., a deactivated microenvironment). In some embodiments, the activation condition refers to an environment comprising a protease capable of cleaving the proteolytically cleavable linker of the binder disclosed herein. In some embodiments, the deactivation condition refers to an environment not comprising a protease capable of cleaving the proteolytically cleavable linker of the binder disclosed herein. In some embodiments, the binder does not cause, or does not cause, an increase in myeloid cell activity and / or target cell death (e.g., direct and / or indirect target cell death, e.g., target cell phagocytosis) under deactivation conditions.

[0209] In some embodiments, the binder, under activation conditions, causes an increase (e.g., a significant increase) in myeloid cell activity and / or target cell death (e.g., direct death and / or indirect death). In some embodiments, the binder causes an increase or level of myeloid cell activity and / or target cell death (e.g., direct death and / or indirect death) under activation conditions that is at least 10% greater than (e.g., at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least twice as much, at least three times as much, at least four times as much, at least five times as much, at least ten times as much, at least 100 times as much, at least 1,000 times as much, at least 10,000 times as much, at least 50,000 times as much, at least 100,000 times as much, at least 1,000,000 times as much, or more) than under deactivation conditions.

[0210] In some embodiments, the affinity of an APP-binding domain not bound to a shielding domain (e.g., under activation conditions) to its target APP is at least 20% greater than the affinity of an APP-binding domain bound to a shielding domain (e.g., under deactivation conditions). In some embodiments, the affinity of an APP-binding domain not bound to a shielding domain (e.g., under activation conditions) to its target APP is at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least twice as much, at least three times as much, at least four times as much, at least five times as much, at least ten times as much, at least 100 times as much, or at least 1000 times as much as the affinity of an APP-binding domain bound to a shielding domain (e.g., under deactivation conditions).

[0211] In some embodiments, the affinity of an APP-binding domain that is not bound to a shielding domain (for example, under activation conditions) to its target APP may be about 200 nM or less. In some embodiments, the affinity of an APP-binding domain that is not bound to a shielding domain (for example, under activation conditions) to its target APP may be about 500 nM or less, about 450 nM or less, about 400 nM or less, about 350 nM or less, about 300 nM or less, about 250 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 45 nM or less, about 40 nM or less, about 35 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 8 nM or less, about 7.5 nM or less, about 7 nM or less, about 6.5 nM or less, about 6 nM or less, about 5.5 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less lower, about 1 nM or less, about 500 pM or less, about 100 pM or less, about 60 pM or less, about 50 pM or less, about 20 pM or less, about 15 pM or less, about 10 pM or less, about 5 pM or less, about 2 pM or less, or less. In some embodiments, the binding affinity is approximately 500 nM, approximately 450 nM, approximately 400 nM, approximately 350 nM, approximately 300 nM, approximately 250 nM, approximately 200 nM, approximately 100 nM, approximately 50 nM, approximately 30 nM, approximately 20 nM, approximately 10 nM, approximately 7.5 nM, approximately 7 nM, approximately 6.5 nM, approximately 6 nM, approximately 5 nM, approximately 4.5 nM, approximately 4 nM, approximately 3.5 nM, approximately 3 nM, approximately 2.5 nM, approximately 2 nM, approximately 1.5 nM, approximately 1 nM, approximately 500 pM, approximately 100 pM, approximately 50 pM, approximately 20 pM, approximately 10 pM, approximately 5 pM, or approximately 2 pM, or any range between these (e.g., approximately 5 nM to approximately 35 nM). In some embodiments, the affinity of the APP-binding domain that is not bound to the shielding domain (for example, under activation conditions) to its target APP is approximately 1 × 10⁻⁶ -7 M or less (about 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 It may be less than or equal to M, or less than or equal to M. In some embodiments, affinity (K DThis can be determined using assays well known in the art, such as by using surface plasmon resonance (SPR) technology in BIACORE® assay instruments.

[0212] In some embodiments, the affinity (K) of the APP binding domain to the shielding domain (for example, under inactivation conditions) D ) may be about 200 nM or less. In some embodiments, the affinity of the APP-binding domain not bound to the shielding domain (for example, under activation conditions) to the target APP is 500 nM or more, about 450 nM or more, about 400 nM or more, about 350 nM or more, about 300 nM or more, about 250 nM or more, 200 nM or more, about 100 nM or more, about 50 nM or more, about 45 nM or more, about 40 nM or more, about 35 nM or more, about 30 nM or more, about 25 nM or more, about 20 It may be nM or greater, approximately 15nM or greater, approximately 10nM or greater, approximately 8nM or greater, approximately 7.5nM or greater, approximately 7nM or greater, approximately 6.5nM or greater, approximately 6nM or greater, approximately 5.5nM or greater, approximately 5nM or greater, approximately 4nM or greater, approximately 3nM or greater, approximately 2nM or greater, approximately 1nM or greater, approximately 500pM or greater, approximately 100pM or greater, approximately 60pM or greater, approximately 50pM or greater, approximately 20pM or greater, approximately 15pM or greater, approximately 10pM or greater, approximately 5pM or greater, approximately 2pM or greater, or more. In some embodiments, the binding affinity is approximately 500 nM, approximately 450 nM, approximately 400 nM, approximately 350 nM, approximately 300 nM, 250 nM, approximately 200 nM, approximately 100 nM, approximately 50 nM, approximately 30 nM, approximately 20 nM, approximately 10 nM, approximately 7.5 nM, approximately 7 nM, approximately 6.5 nM, approximately 6 nM, approximately 5 nM, approximately 4.5 nM, approximately 4 nM, approximately 3.5 nM, approximately 3 nM, approximately 2.5 nM, approximately 2 nM, approximately 1.5 nM, approximately 1 nM, approximately 500 pM, approximately 100 pM, approximately 50 pM, approximately 20 pM, approximately 10 pM, approximately 5 pM, or approximately 2 pM, or any range greater than or between these (e.g., approximately 5 nM to approximately 35 nM). In some embodiments, the affinity of the APP-binding domain that is not bound to the shielding domain (for example, under activation conditions) to its target APP is approximately 1 × 10⁻⁶ -7 M or more (about 10 -8 M or above, 10 -9 M or above, 10-10 M or above, 10 -11 It may be M or greater, or less than or equal to M. In some embodiments, affinity (K D This can be determined using assays well known in the art, such as by using surface plasmon resonance (SPR) technology in BIACORE® assay instruments.

[0213] In some embodiments, when the APP binding domain is bound by the shielding domain, the APP binding domain has an affinity for its corresponding APP that is reduced by approximately 1x or more, approximately 2x or more, approximately 5x or more, approximately 10x or more, approximately 20x or more, approximately 50x or more, approximately 500x or more, approximately 1000x or more, approximately 1000x or more, approximately 5000x or more, or more, or any range in between (e.g., approximately 500x to approximately 1000x) compared to when the APP binding domain is not bound by the shielding domain.

[0214] In some embodiments, the shielding domain has a dissociation constant for binding to the APP-binding domain that is greater than the dissociation constant for binding of the APP-binding domain to its corresponding APP. In some embodiments, when the shielding domain is cleaved from the binder by cleavage of a proteolytically cleavable linker, the shielding domain does not interfere with or compete with the binding of the APP-binding domain to its APP.

[0215] In some embodiments, the shielding domain of the binder as encompassed by this disclosure may be an antibody or antibody fragment that binds to the APP binding domain of the binder. In some embodiments, the shielding domain of the binder as encompassed by this disclosure may be a receptor protein that binds to the APP binding domain of the binder. In some embodiments, the shielding domain of the binder as encompassed by this disclosure may be a fragment or domain of a receptor protein that binds to the APP binding domain of the binder. In some embodiments, the shielding domain of the binder as encompassed by this disclosure may be a receptor ligand that binds to the APP binding domain of the binder. In some embodiments, the shielding domain of the binder as encompassed by this disclosure may be a fragment or domain of a receptor ligand that binds to the APP binding domain of the binder. In some embodiments, the shielding domain of the binder as encompassed by this disclosure may be a small molecule that binds to the APP binding domain of the binder. In some embodiments, the shielding domain of the binder as encompassed by this disclosure may be an aptamer that binds to the APP binding domain of the binder.

[0216] In some embodiments, the shielding domain binds to the APP-binding domain, which is a receptor protein. In some embodiments, the shielding domain binds to the APP-binding domain, which is a fragment or domain of a receptor protein. In some embodiments, the shielding domain binds to the APP-binding domain, which is a receptor ligand. In some embodiments, the shielding domain binds to the APP-binding domain, which is a fragment or domain of a receptor ligand. In some embodiments, the shielding domain binds to the APP-binding domain, which is an antibody or antibody fragment. In some embodiments, the shielding domain binds to the APP-binding domain, which is a small molecule. In some embodiments, the shielding domain binds to the APP-binding domain, which is an aptamer.

[0217] In some embodiments, the shielding domain binds to the APP-binding domain, which is the SIRPα protein. In some embodiments, the shielding domain binds to the APP-binding domain, which is a fragment or domain of the SIRPα protein. In some embodiments, the shielding domain binds to the APP-binding domain, which is the SIGLEC10 protein. In some embodiments, the shielding domain binds to the APP-binding domain, which is a fragment or domain of the SIGLEC10 protein. In some embodiments, the shielding domain binds to the APP-binding domain, which is the PD-1 protein. In some embodiments, the shielding domain binds to the APP-binding domain, which is a fragment or domain of the PD-1 protein. In some embodiments, the shielding domain binds to the APP-binding domain, which is the LILRB1 protein. In some embodiments, the shielding domain binds to the APP-binding domain, which is a fragment or domain of the LILRB1 protein.

[0218] In some embodiments, the shielding domain binds to an APP-binding domain which may contain at least one immunoglobulin heavy chain and / or at least one immunoglobulin light chain. In some embodiments, the shielding domain binds to an APP-binding domain which may contain at least one immunoglobulin heavy chain variable domain and / or at least one immunoglobulin light chain variable domain. In some embodiments, the shielding domain binds to an APP-binding domain which may contain CDR1, CDR2, and CDR3 of at least one immunoglobulin heavy chain variable domain and / or CDR1, CDR2, and CDR3 of at least one immunoglobulin light chain variable domain.In some embodiments, the shielding domain that binds to the APP binding domain is the extracellular domain (ECD) of a cell surface expression protein, a variant version of the ECD of a cell surface expression protein manipulated to improve binding to the target, an intrabody, a domain antibody, an antibody mime, Zybody®, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd' fragment, an Fd fragment, an isolated CDR or a set thereof, a single-chain antibody, a single-chain Fv (scFv), a disulfide-linked Fv (sdFv), a polypeptide-Fc fusion, a single-domain antibody (e.g., a shark single-domain antibody (IgNAR, etc.) or a fragment thereof), a camel antibody, a camelized antibody, a shielding antibody (e.g., Probody®), an affibody, an anti-idiotype (anti-Id) antibody (e.g., This may include, or contain, any antigen-binding fragment of any of the above, including anti-anti-Id antibodies, single-stranded or tandem diabodies (TandAb®), VHH, Anticalin®, Nanobody® minibodies, BiTE®, Ankyrin repeat proteins or DARPIN®, Avimer®, DART, TCR-like antibodies, Adnectin®, Affilin®, Trans-body®, Affibody®, TrimerX®, Microprotein, Fynomer®, Centyrin®, KALBITOR®, CAR, engineered TCRs, or any of the above.

[0219] 1. SIRPα binding domain This specification provides SIRPα-binding domains. In some embodiments, the SIRPα-binding domain can be used as a shielding domain in the provided constructs. In the embodiments herein, the SIRPα-binding domain is a VHH-containing molecule comprising at least one VHH domain that specifically binds to SIRPα. In some embodiments, the VHH domain binds to human SIRPα. In some embodiments, the VHH domain binds to one or both alleles of wild-type human SIRPα. In some embodiments, the VHH domain binds to variant SIRPα that includes one or more amino acid modifications compared to wild-type SIRPα. In some embodiments, the VHH domain binds to variant SIRPα that has a higher affinity for CD47 than wild-type SIRPα.

[0220] Wild-type SIRPα and various manipulated variants of SIRPα are known and can be used as APP-binding domains in the binding constructs described herein, including any of those described in Section II.A. In some embodiments, the VHH domain binds to high-affinity variant CV1 SIRPα (as described in Weiskopf K, et al. Science. 2013 and Ho CC, et al. JBC. 2015; corresponding to the mutations V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I based on the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). In some embodiments, the VHH domain binds to variant SIRPα which has a higher affinity for CD47 than wild-type SIRPα but a lower affinity than the CV1 SIRPα variant. In some embodiments, the VHH domain binds to variant SIRPα having the E54Q mutation. In some of the embodiments provided, the VHH domain is panreactive and can bind to the wild-type SIRPα allele and one or more variants (including CV1 or variants having the E54Q mutation). In some of the embodiments provided, the VHH domain binds to SIRPα having the sequence shown in any one of SEQ ID NOs: 10, 27, 99, or 109. In some of the embodiments provided, the VHH domain binds to SIRPα having the sequence shown in any one of SEQ ID NOs: 103, 104, 105, or 108.

[0221] In some embodiments, the VHH domain is an antibody fragment that is a single monomeric variable antibody domain capable of selectively binding to a specific antigen. With a molecular weight of only 12–15 kDa, the VHH domain (also called a single-domain antibody) is much smaller than a typical antibody (150–160 kDa) which consists of two heavy protein chains and two light chains, and is even smaller than a Fab fragment (approximately 50 kDa, one light chain and half a heavy chain) and a single-chain variable fragment (approximately 25 kDa, two variable domains (one derived from the light chain and one from the heavy chain)).

[0222] A single-domain antibody is an antibody in which the complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies that naturally lack a light chain, single-domain antibodies derived from conventional quadruple-chain antibodies, engineered antibodies, and single-domain skeletons other than those derived from antibodies. Single-domain antibodies may be derived from any species (including, but not limited to, mice, humans, camels, llamas, alpacas, vicuñas, guanacos, sharks, goats, rabbits, and / or cattle). In some embodiments, the single-domain antibodies used herein are naturally occurring single-domain antibodies known as light-chain-lacking heavy-chain antibodies. For clarity, this variable domain derived from a light-chain-lacking heavy-chain antibody is referred to herein as VHH to distinguish it from the conventional VH of quadruple-chain immunoglobulins. Such VHH molecules may be derived from antibodies produced in camelid species (e.g., camels, llamas, dromedary camels, alpacas, vicuñas, and guanacos). Other species besides camelids may also produce heavy-chain antibodies that naturally lack light chains, and such VHHs are within the scope of this disclosure.

[0223] Methods for screening VHH domains (including VHH-binding polypeptides having desired specificity for SIRPα) include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), enzyme assays, flow cytometry, and other immunologically mediated techniques known in the art.

[0224] The VHH domains provided herein include SIRPα (human-derived) (such as any of the following).

[0225] In some embodiments, the VHH domain that binds to SIRPα may be derived from a non-human species and may be humanized. Humanized antibodies (such as VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate the human immune response to non-human antibodies, which can cause an immune response to antibody therapeutics and reduce the effectiveness of the therapeutic agent. Generally, humanized antibodies contain one or more variable domains in which the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies optionally also contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues derived from a non-human antibody (e.g., the antibody from which the CDR residue is derived) to restore or improve antibody specificity or affinity, for example.

[0226] Humanized antibodies and their production methods are outlined, for example, in Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633, for example, Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 ("Resurfacing" is described); Dall'Acqua et al. This is further explained in al., (2005) Methods 36:43-60 (which describes "FR shuffling"); and in Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br.J.Cancer, 83:252-260 (which describes the "guided selection" approach to FR shuffling).

[0227] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the “best fit” method (see, e.g., Sims et al. (1993) J.Immunol. 151:2296); framework regions derived from consensus sequences of human antibodies of specific heavy chain variable region subgroups (see, e.g., Carter et al. (1992) Proc.Natl.Acad.Sci.USA, 89:4285 and Presta et al. (1993) J.Immunol, 151:2623); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front.Biosci. 13:1619-1633); and framework regions obtained from screening of FR libraries (e.g., Baca et al. This includes (see al., (1997) J. Biol. Chem. 272:10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, humanized VHH is created by replacing the FR region of VHH with a human FR region. In some embodiments, replacing certain FR residues in human FR improves one or more properties of the humanized VHH. A VHH domain having such replaced residues is still referred to herein as “humanized”.

[0228] This specification provides VHH domains that bind to SIRPα (SIRPα-binding VHH domains or SIRPα VHH domains), the VHH domains comprising CDR1, CDR2, and CDR3 contained in a VHH amino acid sequence selected from any of SEQ ID NOs: 13-21 and 28-36, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a VHH amino acid sequence selected from any one of SEQ ID NOs: 13-21 and 28-36. In some embodiments, the SIRPα VHH domains provided herein include CDR1 represented by any one of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, and 45; CDR2 represented by any one of SEQ ID NOs: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and CDR3 represented by any one of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. The provided SIRPα VHH domains include SIRPα VHH domains having an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 13-21 and 28-36, or a VHH amino acid sequence selected from any one of SEQ ID NOs: 13-21 and 28-36. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in any one of SEQ ID NOs: 13-21 and 28-36.

[0229] In some embodiments, the SIRPα VHH domain provided herein includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with CDR1, CDR2, CDR3, or the VHH amino acid sequence shown in SEQ ID NO: 13, contained within the VHH domain shown in SEQ ID NO: 13. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 13.

[0230] In some embodiments, the SIRPα VHH domain provided herein includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with CDR1, CDR2, CDR3, or the VHH amino acid sequence shown in SEQ ID NO: 14, contained within the VHH domain shown in SEQ ID NO: 14. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 14.

[0231] In some embodiments, the SIRPα VHH domain provided herein includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with CDR1, CDR2, CDR3, or the VHH amino acid sequence shown in SEQ ID NO: 15, contained within the VHH domain shown in SEQ ID NO: 15. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 15.

[0232] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0233] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 17.

[0234] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 18, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 18.

[0235] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 19.

[0236] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 20, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 20.

[0237] In some embodiments, the SIRPα VHH domain provided herein includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with CDR1, CDR2, CDR3, or the VHH amino acid sequence shown in SEQ ID NO: 21, contained within the VHH domain shown in SEQ ID NO: 21. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 21.

[0238] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 28, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 28.

[0239] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 29, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 29.

[0240] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 30, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 30.

[0241] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 31, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 31. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 31. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 31.

[0242] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 32.

[0243] In some embodiments, the SIRPα VHH domain provided herein comprises CDR1, CDR2, or CDR3 contained in the VHH domain set forth in SEQ ID NO: 33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 33.

[0244] In some embodiments, the SIRPα VHH domain provided herein comprises CDR1, CDR2, or CDR3 contained in the VHH domain set forth in SEQ ID NO: 34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 34.

[0245] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 35, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 35.

[0246] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, CDR3 contained in the VHH domain shown in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the VHH amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the SIRPα VHH domain has the amino acid sequence shown in SEQ ID NO: 36.

[0247] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 37, 46, and 61, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 38, 46, and 61, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 39, 47, and 62, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 40, 48, and 63, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 41, 49, and 64, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 37, 50, and 61, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 42, 51, and 65, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 43, 52, and 66, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 37, 53, and 67, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 44, 54, and 68, respectively. In some embodiments, the SIRPα VHH domains provided herein include CDR1, CDR2, and CDR3, as shown in SEQ ID NOs: 43, 55, and 63, respectively. In some embodiments, the SIRPα VHH domains provided herein include CDR1, CDR2, and CDR3, as shown in SEQ ID NOs: 40, 56, and 69, respectively.In some embodiments, the SIRPα VHH domain provided herein comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 37, 57, and 70, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 40, 55, and 63, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 41, 58, and 71, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 43, 59, and 72, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 37, 60, and 73, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 45, 56, and 73, respectively.

[0248] In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 46, and CDR3 shown in SEQ ID NO: 61. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 38, CDR2 shown in SEQ ID NO: 46, and CDR3 shown in SEQ ID NO: 61. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 39, CDR2 shown in SEQ ID NO: 47, and CDR3 shown in SEQ ID NO: 62. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 40, CDR2 shown in SEQ ID NO: 48, and CDR3 shown in SEQ ID NO: 63. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 49, and CDR3 shown in SEQ ID NO: 64. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 50, and CDR3 shown in SEQ ID NO: 61. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 42, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 65. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 43, CDR2 shown in SEQ ID NO: 52, and CDR3 shown in SEQ ID NO: 66. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 53, and CDR3 shown in SEQ ID NO: 67. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 54, and CDR3 shown in SEQ ID NO: 68. In some embodiments, the SIRPα VHH domains provided herein include CDR1 shown in SEQ ID NO: 43, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 63.In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 40, CDR2 shown in SEQ ID NO: 56, and CDR3 shown in SEQ ID NO: 69. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 57, and CDR3 shown in SEQ ID NO: 70. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 40, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 63. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 58, and CDR3 shown in SEQ ID NO: 71. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 43, CDR2 shown in SEQ ID NO: 59, and CDR3 shown in SEQ ID NO: 72. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 60, and CDR3 shown in SEQ ID NO: 73. In some embodiments, the SIRPα VHH domain provided herein includes CDR1 shown in SEQ ID NO: 45, CDR2 shown in SEQ ID NO: 56, and CDR3 shown in SEQ ID NO: 73.

[0249] In some embodiments, the SIRPα VHH domain is used as a shielding domain in any of the provided multispecific antigen-binding constructs, for example, to shield the CD47 APP-binding domain.

[0250] In some embodiments, the SIRPα VHH domain is used as a component of a bispecific macrophage enhancer (BiME). In some embodiments, a bispecific molecule is provided comprising a SIRPα VHH domain that binds to SIRPα and can block the interaction between CD47 on a cell and SIRPα on a phagocytic cell, and a second antigen, and an antibody having bispecificity to SIRPα and the second antigen is called a bispecific macrophage enhancing (BiME) antibody. In some embodiments, such a binding molecule can induce phagocytic death and immune response against target cells (such as tumor cells). In some embodiments, the bispecific binding molecule can crosslink two cells (effector and target) in close proximity, as a result allowing other cell receptors and membrane components on both cells to interact, thereby enabling effector myeloid cells to induce engulfment of target cells.

[0251] In some embodiments, the bispecific antibody is targeted to SIRPα and a second antigen. Therefore, in some cases, the target antibody is a bispecific or multispecific antibody that specifically binds to SIRPα and at least a second antigen. The second antigen may include any tumor-associated antigen. Exemplary tumor-associated antigens include any of those listed in Table 2. Other exemplary second antigens are any cancer cell markers (e.g., CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), EGFR, HER2, CD117, C-Met, PTHR2, HAVCR2 (TIM3)). In some cases, exemplary bispecific antibodies include a SIRPα VHH domain sequence (e.g., CDR) disclosed herein that provides specific binding to SIRPα, and a sequence (e.g., CDR) derived from an antibody that binds to a cancer cell marker. Examples of antibodies having a CDR that provides specific binding to a cancer cell marker include any of those described herein. In some embodiments, antibodies directed to cancer markers are cetuximab (bound to EGFR), panitumumab (bound to EGFR), rituximab (bound to CD20), trastuzumab (bound to HER2), pertuzumab (bound to HER2), alemtuzumab (bound to CD52), or brentuximab (bound to CD30).

[0252] (i) Exemplary characteristics The SIRPα VHH domains provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various known assays.

[0253] In some embodiments, the SIRPα VHH domain has one or more specific functional properties, such as binding properties including binding to SIRPα (wild-type SIRPα and / or variant SIRPα). In some embodiments, the SIRPα VHH domain has the ability to bind to SIRPα with at least a certain affinity. In some embodiments, the provided SIRPα VHH domain binds to a specific epitope (such as epitopes of SIRPα) with moderate to high affinity. In some embodiments, the binding of the SIRPα VHH domain (such as the binding of the SIRPα VHH domain to SIRPα) is measured by one of several known methods. Binding affinity is K D , K A , or EC 50 It can be measured as follows. In some embodiments, affinity is the equilibrium dissociation constant (K D ) is represented by. In some embodiments, affinity is EC 50 It is represented by [this].

[0254] Binding affinity, equilibrium dissociation constant (K D ), equilibrium binding constant (K A ), EC 50 , ON velocity (coupling rate constant; k on or k a ;1 / Ms or M -1 s -1 (units), and off-velocity (dissociation rate constant; k off or k d ; 1 / s or s -1Various assays are known for evaluating the units of binding affinity (of SIRPα) and / or determining whether a binding molecule (e.g., an antibody or a fragment thereof) specifically binds to a particular ligand (e.g., an antigen). The binding affinity of a binding molecule can be determined by using one of several well-known binding assays. For example, in some embodiments, surface plasmon resonance (SPR) analysis can be used with a Carterra® LSA® instrument to determine the binding kinetics and constants of a complex between two proteins (e.g., an antibody or a fragment thereof and an antigen) (see, e.g., Scatchard et al., Ann. NYAcad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993). In one embodiment, the SIRPα VHH domain is tested for SIRPα binding activity by, for example, known methods. In some embodiments, the binding of the SIRPα VHH domain to an antigen such as SIRPα (wild-type SIRPα or variant SIRPα) is evaluated by methods known in the art (such as ELISA, Western blotting, flow cytometry assays, and / or surface plasmon resonance (SPR)).

[0255] In some embodiments, the equilibrium dissociation constant (K) of the SIRPα VHH domain relative to SIRPα is determined to be the same as that of SIRPα. D) refers to 0.1 or approximately 0.1 nM to 10 or approximately 10 μM, 0.1 or approximately 0.1 nM to 50 or approximately 50 nM, 0.1 or approximately 0.1 nM to 40 or approximately 40 nM, 0.1 or approximately 0.1 nM to 30 or approximately 30 nM, 0.1 or approximately 0.1 nM to 20 or approximately 20 nM, 0.1 or approximately 0.1 nM to 10 or approximately 10 nM, 0.1 or approximately 0.1 nM to 1 or approximately 1 nM, 1 or approximately 1 nM to 500 or approximately 500 nM, 1 or approximately 1 nM to 50 or The binding affinity of the VHH domain to SIRPα is approximately 50 nM, 1 or approximately 1 nM to 40 or approximately 40 nM, 1 or approximately 1 nM to 30 or approximately 30 nM, 1 or approximately 1 nM to 20 or approximately 20 nM, 1 or approximately 1 nM to 10 or approximately 10 nM, 10 or approximately 10 nM to 500 or approximately 500 nM, 10 or approximately 10 nM to 50 or approximately 50 nM, 10 or approximately 10 nM to 40 or approximately 40 nM, 10 or approximately 10 nM to 30 or approximately 30 nM, or 10 or approximately 10 nM to 20 or approximately 20 nM. In a particular embodiment, the binding affinity of the VHH domain to SIRPα is (EC 50 ) and / or equilibrium dissociation constant (K D) are 50nM, 40nM, 30nM, 25nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM, or approximately 50nM, 40nM, 30nM, 25nM, 20nM, 19 nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM, or less than 50nM, less than 40nM, less than 30nM, less than 25nM, less than 20nM, less than 19nM, less than 18nM, less than 17nM, 16n The range is defined as less than M, less than 15nM, less than 14nM, less than 13nM, less than 12nM, less than 11nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1nM, or approximately less than 50nM, less than 40nM, less than 30nM, less than 25nM, less than 20nM, less than 19nM, less than 18nM, less than 17nM, less than 16nM, less than 15nM, less than 14nM, less than 13nM, less than 12nM, less than 11nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1nM, or any of the above.

[0256] In some embodiments, the SIRPα VHH domain binds to SIRPα with a binding affinity of less than nanomolar concentration, for example, less than 1 nM or about less than 1 nM (less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM, or about less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM). In some embodiments, the equilibrium dissociation constant (K) of the binding molecule (e.g., the SIRPα VHH domain) to SIRPα (wild-type or variant SIRPα, etc.) is D) are approximately 0.01 nM to approximately 1 μM, 0.1 nM to 1 μM, 1 nM to 1 μM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM to 500 nM, 50 nM to 100 nM, or 100 nM to 500 nM, or approximately 0.0 The ranges are 1 nM to approximately 1 μM, approximately 0.1 nM to 1 μM, approximately 1 nM to 1 μM, approximately 1 nM to 500 nM, approximately 1 nM to 100 nM, approximately 1 nM to 50 nM, approximately 1 nM to 10 nM, approximately 10 nM to 500 nM, approximately 10 nM to 100 nM, approximately 10 nM to 50 nM, approximately 50 nM to 500 nM, approximately 50 nM to 100 nM, or approximately 100 nM to 500 nM. In certain embodiments, the equilibrium dissociation constant (K) of the binding molecule to SIRPα (e.g., the SIRPα VHH domain) is... D) refers to 10 μM, 5 μM m, 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM, or less, or approximately 10 μM, 5 μM m, 1 μM, 500 nM, 100 nM, 50 nM, 4 0nM, 30nM, 25nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM or less, or less than 10μM, less than 5μM, less than 1μM, less than 500nM, less than 100nM, less than 50nM, less than 40nM, less than 30nM, less than 25nM, less than 20nM Full, less than 19nM, less than 18nM, less than 17nM, less than 16nM, less than 15nM, less than 14nM, less than 13nM, less than 12nM, less than 11nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1nM, or less than approximately 10μM, less than 5μM, less than 1μM, less than 500nM, less than 100nM, less than 50nM, 40n The range is defined as less than M, less than 30 nM, less than 25 nM, less than 20 nM, less than 19 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM, or less than that, or any of the above.

[0257] In some embodiments, the provided SIRPα VHH domain binds to SIRPα with a binding affinity that allows the SIRPα VHH domain to spontaneously release from SIRPα after cleavage of a protease-cleavable linker. In some embodiments, to promote the antiphagocytic activity of the provided multispecific binding construct, the koff value of the provided SIRPα VHH domain cannot be too low and must be greater than that of the wild-type SIRPα or variant SIRPα of the construct against wild-type human CD47 (e.g., cell surface-expressed CD47). Furthermore, the wild-type SIRPα or variant SIRPα of the construct should bind to wild-type human CD47 (e.g., cell surface-expressed CD47) faster than the cleaved and dissociated shield can re-bind, and consequently, the kon value of the SIRPα VHH domain should be smaller than that of the wild-type SIRPα or variant SIRPα antigen. In some embodiments, the SIRPα VHH domain has a lower affinity for binding to wild-type SIRPα or variant SIRPα of the construct than for binding to wild-type human CD47 (such as cell surface-expressed CD47), i.e., a larger dissociation constant (K). D ) has. In some embodiments, the SIRPα VHH domain has K for wild-type human CD47 (cell surface-expressed CD47, etc.) or for wild-type human SIRPα or its variants. D A dissociation constant (K) that is at least 2, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times larger than the original. D ) binds to wild-type human SIRPα or its variants. In some embodiments, the K of the SIRPα VHH domain DThe molecular weight is 1 nM or greater. In such embodiments, after cleavage of a protease-cleavable linker (the SIRPα VHH domain is linked to SIRPα via this protease-cleavable linker), the SIRPα VHH domain is released from SIRPα. In such examples, SIRPα no longer binds to the SIRPα VHH domain and can interact with other binding partners (multiple, e.g., CD47).

[0258] C. A linker capable of cleaving protein. This disclosure provides, in particular, a binder comprising a shielding domain linked to a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker is present in a polypeptide comprising multiple domains provided herein (e.g., including at least an APP-binding domain and a shielding domain). In some embodiments, the shielding domain is positioned such that when the proteolytically cleavable linker is cleaved, the shielding domain is separated from at least one other domain or all other domains present in the polypeptide and / or binder. In this configuration, the shielding domain substantially inhibits the binding of the APP-binding domain to the APP target until the proteolytically cleavable peptide linker is cleaved in the desired environment.

[0259] In some embodiments, the proteolytically cleavable linker includes cleavage sites (such as protease cleavage sites that are recognized and can be cleaved by proteases). The proteolytically cleavable linker includes an amino acid sequence that can act as a substrate for proteases (such as extracellular proteases).

[0260] In some embodiments, the proteolytically cleavable linker can be cleaved by a human and / or biologically relevant protease (e.g., a protease expressed by one or more cells and / or tissues of the human body). In some embodiments, the proteolytically cleavable linker can be cleaved by an extracellular protease. In some embodiments, the proteolytically cleavable linker can be cleaved by a cell surface protease. In some embodiments, the proteolytically cleavable linker can be cleaved by an intracellular protease. In some embodiments, the proteolytically cleavable linker can be cleaved by an aminopeptidase. In some embodiments, the proteolytically cleavable linker can be cleaved by an aspartyl protease. In some embodiments, the proteolytically cleavable linker can be cleaved by a metalloprotease. In some embodiments, the proteolytically cleavable linker can be cleaved by a cysteine ​​protease. In some embodiments, the proteolytically cleavable linker can be cleaved by a serine protease. In some embodiments, the proteolytically cleavable linker can be cleaved by a threonine protease.

[0261] In some embodiments, the proteolytically cleavable linkers are ABHD12 (containing the ab hydrolase domain 12), ABHD12B (containing the ab hydrolase domain 12B), ABHD13 (containing the ab hydrolase domain 13), ABHD17A (sequence-similar family 108, member A1), ABHD17B (sequence-similar family 108, member B1), ABHD17C (sequence-similar family 108, member C1), ABHD4 (containing the ab hydrolase domain 4), ABHD5 (CGI-58), ACE (Angiotensin-Converting Enzyme 1), ACE2 (Angiotensin-Converting Enzyme 2), ACE3P (Angiotensin-Converting Enzyme 3), ACR (Acrosin), ACY1 (Aminoacylase), ACY3 (Aspartoacylase-3), ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM1A (ADAM1a), ADAM2 (ADAM2 / Fertilin-b), ADAM20, ADAM21, ADAM22, ADAM23, ADAM25 (Testase 2), ADAM28, ADAM29, ADAM30, ADAM32, ADAM33, ADAM3B(ADAM3B), ADAM4, ADAM4B(ADAM4B), ADAM5, ADAM6, ADAM7, ADAM8, ADAM9, ADAMDEC1(DECYSIN), ADAMTS1, ADAMTS10, AD AMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ADAMTS5(ADAMTS5 / 11), AD AMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADGB (Calpain 7-like), AEBP1 (Afg3-enh-binding protein 1), AFG3L1P (Afg3-like protein 1), AFG3L2 (Afg3-like protein 2), AGA (Glycosyl asparaginase), AGBL2 (ATP / GTP-binding protein-like 2), AGBL3 (ATP / GTP-binding protein-like 3), AGBL4 (ATP / GTP-binding protein-like 4), AGBL5 (ATP / GTP-binding protein-like 5), AGTPBP1 (ATP / GTP-binding protein 1),ALG13 (UDP-N-acetylglucosaminyltransferase subunit), AMZ1 (archaemetzincin-1), AMZ2 (archaemetzincin-2), ANPEP (aminopeptidase N), AOPEP (aminopeptidase O), APEH (acylaminoacyl-peptidase), ASAH1 (acid ceramidase), ASPA (aspartacylase), ASPRV1 (DDI-related protease), ASRGL1 (glycosylasparaginase-2), A STL (Ovastacin), ATG4A (Autofacin-2), ATG4B (Autofacin-1), ATG4C (Autofacin-3), ATG4D (Autofacin-4), ATXN3 (Ataxin-3), ATXN3L (Ataxin-3-like), AZU1 (Azulocidine), BACE1 (Beta-Secretase 1), BACE2 (Beta-Secretase 2), BAP1 (Ubiquitin C-terminal hydrolase BAP1), BLMH (Bleomycin hydrolase), BMP1 (Procollagen C-proteinase), BRCC3 (BRCC36 / BRCA 2-containing complex, sub3), C1R (complement component C1ra), C1RL (complement C1r homolog), C1S (complement component C1sa), C2 (complement component 2), CAD (dihydroorotase), CAPN1 (calpain 1), CAPN10 (calpain 10), CAPN11 (calpain 11), CAPN12 (calpain 12), CAPN13 (calpain 13), CAPN14 (calpain 14), CAPN15 (calpain 15 / Solh protein), CAPN2 (calpain 2), CAPN3 (calpain 3), CAPN5 (calpain 5), CAP N6 (calpain 6), CAPN7 (calpain 7), CAPN8 (calpain 8), CAPN9 (calpain 9), CARD8 (caspase mobilization domain family, member 8), CASP1 (caspase-1), CASP10 (caspase-10), CASP12 (caspase-12), CASP14 (caspase-14), CASP16P (caspase-14-like), CASP1P2 (homolog ICEY), CASP2 (caspase-2), CASP3 (caspase-3), CASP4 (caspase-4 / 11), CASP5 (caspase-5),CASP6 (caspase-6), CASP7 (caspase-7), CASP8 (caspase-8), CASP9 (caspase-9), CELA1 (pancreatic elastase), CELA2A (pancreatic elastase II (IIA)), CELA2B (pancreatic elastase II morphology B), CELA3A (pancreatic endopeptidase E (A)), CELA3B (pancreatic endopeptidase E (B)), CFB (complement factor B), CFD (complement factor D), CFI (complement factor I), CFLAR (casper / FLIP), CLPP (endopeptidase Clp), CMA1 (chymase ), CNDP1 (carnosine dipeptidase 1), CNDP2 (carnosine dipeptidase 2), COPS5 (CSN5 / JAB1), COPS6 (COPS6), CORIN (choline), CPA1 (carboxypeptidase A1), CPA2 (carboxypeptidase A2), CPA3 (carboxypeptidase A3), CPA4 (carboxypeptidase A4), CPA5 (carboxypeptidase A5), CPA6 (carboxypeptidase A6), CPB1 (carboxypeptidase B), CPB2 (carboxypeptidase U), CPD (carnosine dipeptidase 1) Luboxypeptidase D), CPE (Carboxypeptidase E), CPM (Carboxypeptidase M), CPN1 (Carboxypeptidase N), CPO (Carboxypeptidase O), CPQ (Plasma Glu-Carboxypeptidase), CPVL (Yellow-forming Carboxypeptidase L), CPXM1 (Carboxypeptidase X1), CPXM2 (Carboxypeptidase X2), CPZ (Carboxypeptidase Z), CRMP1 (Dihydropyrimidinase-related protein 1), CTRB1 (Chymotrypsin B), CTRC (Chymotrypsin C), CTRL (chymopasin), CTSA (lysosomal carboxypeptidase A), CYLD (cylindroma protein), CYMP (chymosin), DDI1 (DNA damage-inducing protein), DDI2 (DNA damage-inducing protein 2), DERL1 (Der1-like domain family, member 1), DERL2 (Der1-like domain family, member 2), DERL3 (Der1-like domain family, member 3), DESI1 (de-SUMOylated isopeptidase 1), DESI2 (de-SUMOylated isopeptidase 2),DHH (Desert Hedgehog Protein), DNPEP (Aspartylaminopeptidase), DPEP1 (Membrane Dipeptidase), DPEP2 (Membrane Dipeptidase 2), DPEP3 (Membrane Dipeptidase 3), DPP10 (Dipeptidyl Peptidase 10), DPP3 (Dipeptidyl Peptidase III), DPP4 (Dipeptidyl Peptidase 4), DPP6 (Dipeptidyl Peptidase 6), DPP7 (Dipeptidyl Peptidase II), DPP8 (Dipeptidyl Peptidase 8), DPP9 (Dipeptidyl Peptidase 9), DPYS (Dihydro Lopyrimidinase), DPYSL2 (Dihydropyrimidinase-related protein 2), DPYSL3 (Dihydropyrimidinase-related protein 3), DPYSL4 (Dihydropyrimidinase-related protein 4), DPYSL5 (Dihydropyrimidinase-related protein 5), ECE1 (Endothelin-converting enzyme 1), ECE2 (Endothelin-converting enzyme 2), ECEL1 (DINE peptidase), ECT2L (Epithelial cell transformation sequence 2 oncogene-like), EIF3F (Eukaryotic translation initiation factor F3SF), EIF3H (Eukaryotic translation initiation factor F3SH), ELA NE (neutrophil elastase), ENPEP (aminopeptidase A), EPHX1 (epoxide hydrolase), EPHX4 (epoxide hydrolase-related protein), ERAP1 (aminopeptidase PILS), ERAP2 (aminopeptidase MAMS / L-RAP), ERMP1 (endoplasmic reticulum metallopeptidase 1), ESPL1 (separase), F10 (coagulation factor Xa), F11 (coagulation factor XIa), F12 (coagulation factor XIIa), F2 (thrombin), F7 (coagulation factor VIIa), F9 (coagulation factor IXa), FACE1 (FACE-1 / Z MPSTE24), FACE2 (FACE-2 / RCE1), FAM111A (Sequence-Similar Family 111, A), FAM111B (Sequence-Similar Family 111, B), FAP (Seplase), FOLH1 (Glutamate Carboxypeptidase II), FREM1 (Signalase-like 1), FURIN (Furin), GFPT1 (Gln-fructose-6-P Transamidase 1), GFPT2 (Gln-fructose-6-P Transamidase 2), GFPT3 (Gln-fructose-6-P Transamidase 3),GGH (gamma-glutamyl hydrolase), GGT1 (gamma-glutamyltransferase 1), GGT2 (gamma-glutamyltransferase 2), GGT6 (gamma-glutamyltransferase 6), GGT7 (gamma-glutamyltransferase-like 3), GGTLC1 (gamma-glutamyltransferase 5), GGTLC2 (gamma-glutamyltransferase m-3), GZMA (Granzyme A), GZMB (Granzyme B), GZMH (Granzyme H), GZMK (Granzyme K), GZMM (Granzyme M), HABP2 (Hyaluronan-binding ser protease), HATL2 (HAT-like 2), HATL3 (HAT-like 3), HGF (Hepatocyte Growth Factor), HGFAC (HGF Activator), HM13 (Presenilin Homolog 3 / SPP), HP (Haptoglobin-1), HPN (Heptoglobin-1) HSP90AA1 (Heat Shock 90kDa Protein 1, Alpha), HSP90AB1 (Heat Shock 90kDa Protein 1, Beta), HSP90B1 (Heat Shock Protein 90kDa Beta (Grp94), Member 1 / Tumor Rejection Antigen (gp96)), HTRA1 (Osteoblast Serine Protease), HTRA2 (HTRA2), HTRA3 (HTRA3), HTRA4 (HTRA4), IDE (Inthlysine), IHH (Indian Hedgehog Protein), IMMP1L (Mitochondrial Signaling Peptidase), IMMP2L (Mitochondrial Inner Membrane Protease 2), INPP5E (Mitochondrial Processing Protease), JOSD1 (Josephine-1), JOSD2 (Josephine-2), KEL (Kell Blood Group Protein), KHNYN (KHNYN (Containing KH and NYN domains), KLK1 (kallikrein hK1), KLK10 (kallikrein hK10), KLK11 (kallikrein hK11), KLK12 (kallikrein hK12), KLK13 (kallikrein hK13), KLK14 (kallikrein hK14), KLK15 (kallikrein hK15), KLK2 (kallikrein hK2), KLK3 (kallikrein hK3), KLK4 (kallikrein hK4), KLK5 (kallikrein hK5), KLK6 (kallikrein hK6), KLK7 (kallikrein hK7), KLK8 (kallikrein hK8), KLK9 (kallikrein hK9), KLKB1 (plasma kallikrein), KLKBL3 (plasma kallikrein-like 3), LACTB (beta-lactamase), LAP3 (leucylaminopeptidase), LGMN (regmine), LMLN (leishmanolsin-2), LNPEP (leucyl-cystinylaminopeptidase), LOC440434 (cytozolaranylaminopeptidase-like 1), LONP1 (PIM1 endopeptidase), LONP2 (PIM2 endopeptidase), LPA (apolipoprotein) (a)) LTA4H (leukotriene A4 hydrolase), LTF (lactotransferrin; lactoferrin), LVRN (aminopeptidase Q), MALT1 (paracaspase), MASP1 (MASP1 / 3), MASP2 (MASP2), MASTIN (mastine), MBTPS1 (cyto-1 protease), MBTPS2 (S2P protease), MEP1A (meprin alpha subunit), MEP1B (meprin beta subunit), MEST (mesoderm-specific transcript), METAP1 (methionylaminopeptidase I), M ETAP1D (MAP1D Methionyl Amino Peptidase 1D), METAP2 (Methionyl Amino Peptidase II), MIPEP (Mitochondrial Intermediate Peptidase), MME (Neprilysin), MMEL1 (Neprilysin-2), MMP1 (Collagenase 1), MMP10 (Stromelysin 2), MMP11 (Stromelysin 3), MMP12 (Macrophage Elastase), MMP13 (Collagenase 3), MMP14 (MT1-MMP), MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MM P), MMP19 (MMP19), MMP2 (Gelatinase A), MMP20 (Enamelicin), MMP21 (MMP21), MMP23A (MMP23A), MMP23B (MMP23B), MMP24 (MT5-MMP), MMP25 (MT6-MMP), MMP26 (Matrilysin-2), MMP27 (MMP27), MMP28 (Epiricin), MMP3 (Stromelysin 1), MMP7 (Matrilysin), MMP8 (Collagenase 2), MMP9 (Gelatinase B), MPND (MPND), MST1 (Macrophage-stimulating protein),MYSM1 (MYSM1), N4BP1 (NEDD4-binding protein 1), NAALAD2 (NAALADASE II), NAALADL1 (NAALADASE-like protein 1), NAALADL2 (NAALADASE-like protein 2), NAPSA (Napsin A), NAPSB (Napsin B), NLN (Neurolysin), NLRP1 (NLRP1 self-cleaving protein), NPEPL1 (Aminopeptidase-like protein 1), NPEPPS (Cytosollalanylaminopeptidase), NRD1 (Nardilidine), NRIP2 (Nuclear receptor-interacting protein 2), NRIP3 (Nuclear receptor-interacting protein 3), NSMF (Nasal embryonic LHRH factor), N UP98 (Nucleoporin 98), NYNRIN (containing NYN domain and retroviral integrase), OMA1 (OMA1), OSGEP (O-Sialoglycoprotein endopeptidase), OSGEPL1 (O-Sialoglycoprotein endopeptidase-like 1), OTUB1 (Otsubine-1), OTUB2 (Otsubine-2), OTUD1 (OTU domain-containing 1), OTUD3 (OTU domain-containing 3), OTUD4 (Hin-1 / OTU domain-containing 4), OTUD5 (OTU domain-containing 5), OTU D6A (OTU domain-containing 6A), OTUD6B (OTU domain-containing 6B), OTUD7A (Cezanne-2), OTUD7B (Cezanne / OTU domain-containing 7B), OVCH1 (ovoxymase-like), OVCH2 (ovidactin-like / ovoxymase-2), PA2G4 (growth-related protein 1), PAMR1 (protein C-like), PAN2 (USP52), PAPPA (pappalysin-1), PAPPA2 (pappalysin-2), PARK7 (DJ-1), PARL (presenilin-related rhomboid-like), PCSK 1 (Proprotein convertase 1), PCSK2 (Proprotein convertase 2), PCSK4 (Proprotein convertase 4), PCSK5 (Proprotein convertase 5), PCSK6 (PACE4 proprotein convertase), PCSK7 (Proprotein convertase 7), PCSK9 (Proprotein convertase 9), PEPD (X-prodipeptidase), PGA3 / 4 / 5 (Pepsin A), PGC (Pepsin C), PGPEP1 (Pyroglutamyl-peptidase I), PGPEP1L (Pyroglutamyl-peptidase II),PHEX (PHEX endopeptidase), PIDD1 (PIDD self-processing protein unit 1), PIGK (hGPI8), PIP (GCDFP15), PITRM1 (pitrylysine metalloproteinase 1), PLAT (t-plasminogen activator), PLAU (u-plasminogen activator), PLG (plasminogen), PM20D2 (PM20D2 peptidase), PMPCB (mitochondrial processing peptidase β-subunit), PPAT (Gln-PRPP amide transferase), PPNX (Ppnx), PRCP (lysosomal pro-X) C-peptidase), PREP (prolyl oligopeptidase), PREPL (prolyl oligopeptidase-like), PROC (protein C), PROZ (protein Z), PRPF8 (PRPF8), PRSS1 (cationic trypsin), PRSS12 (neurotrypsin), PRSS16 (thymus-specific serine peptidase), PRSS2 (anionic trypsin(II)), PRSS21 (thymus-specific serine peptidase). Testisin, PRSS22 (cerebral serine proteinase 2), PRSS23 (umbilical vein proteinase), PRSS27 (malapsin), PRSS29P (implantation serine protease 2), PRSS3 (mesotrypsin), PRSS30P (intestinal serine protease 1), PRSS33 (tryptase homolog 2 / EOS), PRSS35 (SPUVE-like), PRSS36 (polycement PRSS37 (Trypsin X2), PRSS38 (Malapsin 2), PRSS3P2 (Trypsin C), PRSS41 (Tryptase homolog 3), PRSS42 (Testicular serine protease 2), PRSS45 (Testicular serine protease 5), PRSS48 (Epidermal-specific SP-like), PRSS50 (Testicular-specific protein tsp50), PRSS53 (Polycellase-3), PRSS54 (Plasma kallikrein-like 4), PRSS55 (Plasma kallikrein-like 2), PRSS56 (Protease, serine, 56), PRSS57 (Complement factor D-like), PRSS8 (Prostasin), PRTN3 (Proteinase 3), PSEN1 (Presenilin 1), PSEN2 (Presenilin 2), PSMA1 (Proteasome alpha-1 subunit), PSMA2 (Proteasome alpha-2 subunit),PSMA3 (proteasome alpha-3 subunit), PSMA4 (proteasome alpha-4 subunit), PSMA5 (proteasome alpha-5 subunit), PSMA6 (proteasome alpha-6 subunit), PSMA7 (Proteasome Alpha 7 subunit), PSMA8 (Proteasome Alpha 8 subunit), PSMB1 (Proteasome Beta 1 subunit), PSMB10 (Proteasome Catalyst subunit 2i), PSMB11 (Proteasome b subunit LMP7-like), PSMB2 (Proteasome Beta 2 subunit), PSMB3 (Proteasome Beta 3 subunit), PSMB4 (Proteasome Beta 4 subunit), PSMB5 (Proteasome Catalyst subunit 3), PSMB6 (Proteasome Catalyst subunit Unit 1), PSMB7 (Proteasome catalytic subunit 2), PSMB8 (Proteasome catalytic subunit 3i), PSMB9 (Proteasome catalytic subunit 1i), PSMD14 (POH1 / PSMD14), PSMD7 (PSMD7), QPCT (Glutaminil cyclase), QPCTL (Glutaminil cyclase 2), RBP3 (Retinol-binding protein 3), RELN (Reelin), REN (Renin), RHBDD1 (Rhomboid domain 1), RHBDD2 (Rhomboid domain 2), RHBDF1 (Rhomboid 5) Homolog 1), RHBDF2 (Rhomboid 5 homolog 2), RHBDL1 (Rhomboid-like protein 1), RHBDL2 (Rhomboid, pulse-like 2), RHBDL3 (Rhomboid, pulse-like 3), RNPEP (Aminopeptidase B), RNPEPL1 (Aminopeptidase B-like 1), SCPEP1 (Serine carboxypeptidase 1), SCRN1 (Cesernin-1), SCRN2 (Cesernin-2), SCRN3 (Cesernin-3), SEC11A (Signalase 18kDa component), SEC11C (Signalase 21kDa component), SENP1 (Sen Trin / SUMOprotease 1), SENP2 (Centrin / SUMOprotease 2), SENP3 (Centrin / SUMOprotease 3), SENP5 (Centrin / SUMOprotease 5), SENP6 (Centrin / SUMOprotease 6), SENP7 (Centrin / SUMOprotease 7), SENP8 (Centrin / SUMOprotease 8), SHH (Sonic Hedgehog Protein), SPG7 (Parapregine), SPPL2A (Presenilin homolog 5), SPPL2B (Presenilin homolog 4 / SPPL2B),SPPL2C (presenilin homolog 2), SPPL3 (presenilin homolog 1 / SPPL3), SPRTN (SprT-like N-terminal domain), ST14 (matryptase), STAMBP (AMSH / STAMBP), STAMBPL1 (AMSH-LP / STAMBPL1), SUPT16H (inhibitor of Ty16 homolog), TAF2 (TBP-related factor 2), TASP1 (taspase), TESP2 (TESP2), TESP3 (TESP3), TESSP3 (testicular serine protease 3), TESSP4 (testicular serine protease 4), TES SP6 (testicular serine protease 6), TFR2 (transferrin receptor 2 protein), TFRC (transferrin receptor protein), THOP1 (thimet oligopeptidase), TINAG (tubulointerstitial nephritis antigen), TINAGL1 (TINAG-related protein), TLL1 (mammalian toroid-like protein 1), TLL2 (mammalian toroid-like protein 2), TMPRSS11A (TMPRSS11A), TMPRSS11B (HAT-like 5), TMPRSS11D (airway trypsin-like protease), TMPRSS11E (DE SC1 protease), TMPRSS11F (HAT-like 4), TMPRSS12 (HAT-related protease), TMPRSS13 (membrane-type mosaic ser protease), TMPRSS15 (enteropeptidase), TMPRSS2 (epitheliasin), TMPRSS3 (transmembrane ser protease 3), TMPRSS4 (transmembrane ser protease 4), TMPRSS5 (spinesin), TMPRSS6 (matryptase-2), TMPRSS7 (matryptase-3), TMPRSS9 (poly Celase-I), TNFAIP3 (A20, TNFα-inducing protein 3), TPP1 (tripeptidyl peptidase I), TPP2 (tripeptidyl peptidase II), TPSAB1 (tryptase alpha / beta 1), TPSB2 (tryptase beta 2), TPSD1 (tryptase delta 1), TPSG1 (tryptase gamma 1), TRAP1 (heat shock protein 75), TRHDE (TRH degrading enzyme), TRY10 (trypsin 10), TRY15 (trypsin 15), TTC28 (HetF-like), TTR (transthyretin),TYSND1 (Arabidopsis thaliana Ser protease-like), UCHL1 (ubiquitin C-terminal hydrolase 1), UCHL3 (ubiquitin C-terminal hydrolase 3), UCHL5 (ubiquitin C-terminal hydrolase 5), UFSP1 (Ufm-1 specific protease 1), UFSP2 (Ufm-1 specific protease 2), UQCRC1 (UCR1), UQCRC2 (UCR2), USP1 (USP1), USP10 (USP10), USP11 (USP11), USP12 (USP12), USP13 (USP13), USP14 (USP14), USP1 5 (USP15), USP16 (USP16), USP17L2 (USP17-like), USP17L9P (USP17), USP18 (USP18), USP19 (USP19), USP2 (USP2), USP20 (USP20), USP21 (USP21), USP22 (USP22), USP24 (USP24), USP25 (USP25), USP26 (USP26), USP27X (USP27), USP28 (USP28), USP29 (USP29), USP3 (USP3), USP30 (USP30), USP31 (USP31), US P32 (NY-REN-60), USP33 (VDU1), USP34 (USP34), USP35 (USP35), USP36 (USP36), USP37 (USP37), USP38 (HP43.8KD), USP39 (SAD1), USP4 (USP4), USP40 (USP40), USP41(USP41), USP42(USP42), USP43(USP43), USP44(USP44), USP45(USP45), USP46(USP46), USP47(USP47), USP48(USP48), USP49(USP49) USP5 (USP5), USP50 (USP50), USP51 (USP51), USP53 (USP53), USP54 (USP54), USP6 (USP6), USP7 (USP7), USP8 (USP8), USP9X (USP9X), USP9Y (USP9Y), USPL1 (ubiquitin-specific peptidase-like 1), VCPIP1 (VCP(p97) / p47 interacting protein), XPNPEP1 (aminopeptidase P1), XPNPEP2 (X-prolylaminopeptidase 2), XPNPEP3 (aminopeptidase P homolog),It may contain motifs that are cleaved and / or cleaved by human proteases selected from XRCC6BP1 (ATP23 peptidase), YME1L1 (YME1-like 1), YOD1 (OTUD2 / YOD1), ZC3H12A (zinc finger CCCH type 12A), ZC3H12B (zinc finger CCCH type 12B), ZC3H12C (zinc finger CCCH type 12C), ZC3H12D (zinc finger CCCH type 12D), ZRANB1 (TRAF-binding protein domain), and / or ZUP1 (zinc finger-containing ubiquitin peptidase 1).

[0262] In some embodiments, the proteolytically cleavable linker may include a motif that is cleaved and / or cleaved by a human protease selected from renin, cathepsin D, cathepsin E, pepsin C, or napsin A, matrix metalloproteinase (MMP), matryptase, urokinase-type plasminogen activator (uPA), disintegrin and metalloproteinase (ADAM), disintegrin and metalloproteinase having a thrombospondin motif (ADAMTS), legmine, urokinase, or hepsin.

[0263] Many representative examples of linker sequences that can be cleaved proteolytically are known in the art, and assays for determining protease-mediated protein sequence cleavage are also well known in the art (see, for example, U.S. Patent 10,259,845 and U.S. Patent Publication 2020 / 0115461).

[0264] In some embodiments, the proteolytically cleavable linker includes a protease cleavage site, which is a tumor-associated protease cleavage site recognized by a protease whose expression is specific to or upregulated in tumor cells or their tumor cell environment. The cleavable linker may be selected based on a protease produced by a tumor in close proximity to cells expressing the target, and / or a protease produced by a tumor that co-localizes in the tissue with the desired target of the multispecific polypeptide construct. Increased levels of proteases with known substrates have been reported in the literature in some cancers (e.g., solid tumors). See, for example, La Rocca et al, (2004) British J. of Cancer 90(7):1414-1421.

[0265] ABHD12、ADAM12、ABHD12B、ABHD13、ABHD17A、A DAM19、ADAM20、ADAM21、ADAM28、ADAM30、ADAM33、ADAM8、ABHD17A、ADAMDEC 1、ADAMTS1、ADAMTS10、ADAMTS12、ADAMTS13、ADAMTS14、ADAMTS15、ADAMTS16、ADAMTS17、ADAMTS18、ADAMTS19、ADAMTS2、ADAMTS20、ADAMTS3、ADAMTS4、A BHD17B、ADAMTS5、ADAMTS6、ADAMTS7、ADAMTS8、ADAMTS9、ADAMTSL1、ADAMTSL2、ADAMTSL3、ABHD17C、ADAMTSL5、ASTL、BMP1、CELA1、CELA2A、CELA2B、CELA 3A、CELA3B、ADAM10、ADAM15、ADAM17、ADAM9、ADAMTS4、CTSE、CTSF、ADAMTSL 4、CMA1、CTRB1、CTRC、CTSO、CTR1、CTSA、CTSW、CTSB、CTSC、CTSD、ESP1、CTSG、 CTSH、GZMA、GZMB、GZMH、CTSK、GZMM、CTSL、CTSS、CTSV、CTSZ、HTRA4、KLK10、 KLK11、KLK13、KLK14、KLK2、KLK4、DPP4、KLK6、KLK7、KLKB1、ECE1、ECE2、ECE L1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP2 3B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1, MMP1 0、PLAT、PLAU、PLG、PRSS1、PRSS12、PRSS2、PRSS21、PRSS3、PRSS33、PRSS4、P RSS55、PRSS57、MMP12、PRSS8、PRSS9、PRTN3、MMP13、MMP14、ST14、TMPRSS10、is a cleavage site recognized by one or more enzymes selected from the group consisting of TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1.,

[0266] In some embodiments, the protease cleavage site is a cleavage site recognized by one or more enzymes selected from the group consisting of ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3.

[0267] In some embodiments, the protease cleavage site is a matrix metalloprotease (MMP) cleavage site, a disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site, a prostate specific antigen (PSA) protease cleavage site, a urokinase-type plasminogen activator (uPA) protease cleavage site, a membrane-type serine protease 1 (MT-SP1) protease cleavage site, a matriptase protease cleavage site (ST14), or a legumain protease cleavage site. In some embodiments, the matrix metalloprotease (MMP) cleavage site is an MMP9 cleavage site, an MMP13 cleavage site, or an MMP2 cleavage site. In some embodiments, the disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site is an ADAM9 metalloprotease cleavage site, an ADAM10 metalloprotease cleavage site, or an ADAM17 metalloprotease cleavage site.

[0268] In some embodiments, the proteolytically cleavable linker is a cleavable peptide. In some embodiments, the cleavable peptide is a 5-mer (i.e., a peptide with an amino acid length of 5), a 6-mer (i.e., a peptide with an amino acid length of 6), a 7-mer (i.e., a peptide with an amino acid length of 7), an 8-mer (i.e., a peptide with an amino acid length of 8), a 9-mer (i.e., a peptide with an amino acid length of 9), a 10-mer (i.e., a peptide with an amino acid length of 10), an 11-mer (i.e., a peptide with an amino acid length of 11), a 12-mer (i.e., a peptide with an amino acid length of 12), a 13-mer (i.e., a peptide with an amino acid length of 13), a 14-mer (i.e., a peptide with an amino acid length of 14), a 15-mer (i.e., a peptide with an amino acid length of 15), a 16-mer (i.e., a peptide with an amino acid length of 16), a 17-mer (i.e., a peptide with an amino acid length of 17), or an 18-mer (i.e., a peptide with an amino acid length of 18).

[0269] In some embodiments, the cleavable linker contains a substrate recognition site or cleavage site of a specific protease (a sequence recognized by the protease's active site and cleaved by the protease). Typically, for example, for serine proteases, the cleavage sequence consists of P1-P4 and P1'-P4' amino acids in the substrate, and the cleavage occurs after the P1 position. Typically, the cleavage sequence of a serine protease is 6 residues long to accommodate the extended substrate specificity of many proteases, but can be longer or shorter depending on the protease. Typically, the cleavable linker contains a cleavable binding sequence P1-P1' recognized by the protease. In some embodiments, the cleavable linker is manipulated, for example, by introducing a substrate recognition site sequence or cleavage sequence of a protease, so that a peptide bond cleavable by a specific protease is introduced.

[0270] In some embodiments, the protease is granzyme B, matryptase, or MMP (such as MMP-2). In some embodiments, the cleavable linker includes a combination of two or more substrate sequences. In some embodiments, each substrate sequence is cleaved by the same protease. In some embodiments, at least two of the substrate sequences are cleaved by different proteases.

[0271] In some embodiments, the cleavable linker contains an amino acid that is a substrate of granzyme B. In some embodiments, the linker cleavable with granzyme B contains an amino acid sequence having the general formula P4P3P2P1↓P1' (SEQ ID NO: 214), where P4 is an amino acid I, L, Y, M, F, V, or A; P3 is an amino acid A, G, S, V, E, D, Q, N, or Y; P2 is an amino acid H, P, A, V, G, S, or T; P1 is an amino acid D or E; and P1' is an amino acid I, L, Y, M, F, V, T, S, G, or A. In some embodiments, the linker cleavable with Granzyme B contains an amino acid sequence having the general formula P4P3P2P1↓P1' (SEQ ID NO: 260), where P4 is amino acid I or L, P3 is amino acid E, P2 is amino acid P or A, P1 is amino acid D, and P1' is amino acid I, V, T, S, or G.

[0272] In some embodiments, the substrate of granzyme B includes the amino acid sequence LEAD (SEQ ID NO: 215), LEPG (SEQ ID NO: 216), or LEAE (SEQ ID NO: 217). In some embodiments, the cleavable linker includes the amino acid sequence IEPDI (SEQ ID NO: 218), LEPDG (SEQ ID NO: 219); LEADT (SEQ ID NO: 220), IEPDG (SEQ ID NO: 221), IEPV (SEQ ID NO: 222), IEPDS (SEQ ID NO: 223), IEPDT (SEQ ID NO: 224), IEPDP (SEQ ID NO: 225), LEPDG (SEQ ID NO: 226), or LEADG (SEQ ID NO: 227).

[0273] In some embodiments, the cleavable linker contains an amino acid that is a substrate of the matryptase. In some embodiments, the cleavable linker contains the sequence P4QAR↓(A / V)(SEQ ID NO: 228), where P4 is any amino acid. In some embodiments, the cleavable linker contains the sequence RQAR(A / V)(SEQ ID NO: 229). In some embodiments, the substrate of the matryptase contains the amino acid sequence RQAR(SEQ ID NO: 230). In some embodiments, the cleavable linker contains the amino acid sequence RQARV(SEQ ID NO: 231).

[0274] In some embodiments, the cleavable linker contains amino acids that are substrates for one or more matrix metalloproteinases (MMPs). In some embodiments, the MMP is MMP-2. In some embodiments, the cleavable linker contains the general formula P3P2P1↓P1' (SEQ ID NO: 232), where P3 is P, V, or A, P2 is Q or D, P1 is A or N, and P1' is L, I, or M. In some embodiments, the cleavable linker contains the general formula P3P2P1↓P1' (SEQ ID NO: 261), where P3 is P, P2 is Q or D, P1 is A or N, and P1' is L or I. In some embodiments, the substrate of the MMP contains the amino acid sequence PAGL (SEQ ID NO: 233).

[0275] In some embodiments, the cleavable linker includes a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for matryptase. In some embodiments, the cleavable linker includes a combination of the amino acid sequence LEAD (SEQ ID NO: 215) and the amino acid sequence RQAR (SEQ ID NO: 230).

[0276] In some embodiments, the cleavable linker includes a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for MMP. In some embodiments, the cleavable linker includes a combination of the amino acid sequence LEAD (SEQ ID NO: 215) and the amino acid sequence PAGL (SEQ ID NO: 233).

[0277] In some embodiments, the cleavable linker includes a combination of an amino acid sequence that is a substrate for matryptase and an amino acid sequence that is a substrate for MMP. In some embodiments, the cleavable linker includes a combination of the amino acid sequence RQAR (SEQ ID NO: 230) and the amino acid sequence PAGL (SEQ ID NO: 233).

[0278] In some embodiments, the cleavable linker includes a combination of an amino acid sequence that is a substrate for granzyme B, an amino acid sequence that is a substrate for matryptase, and an amino acid sequence that is a substrate for MMP. In some embodiments, the cleavable linker includes a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for MMP. In some embodiments, the cleavable linker includes a combination of amino acid sequences LEAD (SEQ ID NO: 215), RQAR (SEQ ID NO: 230), and PAGL (SEQ ID NO: 233).

[0279] The cleavable linkers may include any known linkers. Examples of cleavable linkers are described in Be'liveau et al. (2009) FEBS Journal, 276, U.S. Publication Nos. US20160194399, US20150079088, US20170204139, US20160289324, US20160122425, US20150087810, US20170081397, and U.S. Patent No. US9644016. Non-limiting cleavable peptide sequences are listed in Table 1 of U.S. Patent No. 11,053,294, which is incorporated in its entirety by reference.

[0280] In some embodiments, the severable linker is TGLEADGSPAGLGRQARVG (SEQ ID NO: 234), TGLEADGSRQARVGPAGLG (SEQ ID NO: 235), TGSPAGLEADGSRQARVGS (SEQ ID NO: 236), TGPAGLGLEADGSRQARVG (SEQ ID NO: 237), TGRQARVGLEADGSPAGLG (SEQ ID NO: 238), TGSRQARVGPAGLEADGS (SEQ ID NO: 239), and TGPAGLGSRQARVGLEADGS (SEQ ID NO: 240), GPAGLGLEPDGSRQARVG (SEQ ID NO: 241), GGSGGGGIEPDIGGSGGS (SEQ ID NO: 242), GGSGGGGLEADTGGSGGS (SEQ ID NO: 243), GSIEPDIGS (SEQ ID NO: 244), GSLEA It contains an amino acid sequence selected from the group consisting of DTGS (SEQ ID NO: 245), GGSGGGGIEPDGGGSGGS (SEQ ID NO: 246), GGSGGGGIEPDVGGSGGS (SEQ ID NO: 247), GGSGGGGIEPDSGGSGGS (SEQ ID NO: 248), GGSGGGGIEPDTGGSGGS (SEQ ID NO: 249), GGGSLEPDGSGS (SEQ ID NO: 250), and GPAGLGLEADGSRQARVG (SEQ ID NO: 251), GGEGGGGSGGSGGGS (SEQ ID NO: 252), GSSAGSEAGGSGQAGVGS (SEQ ID NO: 253), GGSGGGGLEAEGSGGGGS (SEQ ID NO: 254), GGSGGGGIEPDPGGSGGS (SEQ ID NO: 255), and TGGSGGGGIEPDIGGSGGS (SEQ ID NO: 256).

[0281] In some embodiments, the proteolytically cleavable linker is the cleavable peptide VHMPLGFLGPRQARVVN (SEQ ID NO: 22). In some embodiments, the proteolytically cleavable linker is the cleavable peptide ISSGLLSGRSDNH (SEQ ID NO: 12).

[0282] In some embodiments, additional linker sequences may be present at the N-terminus and / or C-terminus of the protease-cleavable linker. In some embodiments, an additional linker sequence is present at the N-terminus of the protease-cleavable linker. In some embodiments, an additional linker sequence is present at the C-terminus of the protease-cleavable linker. In some embodiments, an additional linker sequence is present at the N-terminus of the protease-cleavable linker, and an additional linker sequence is also present at the C-terminus of the protease-cleavable linker. The additional linker sequence(s) are typically mobile linker sequences. Particularly preferred linker sequences primarily consist of amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, the linker may contain at least 75% (calculated based on the total number of residues present in the peptide linker) (at least 80%, at least 85%, or at least 90%) of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may consist only of Gly, Ser, Ala, and / or Thr residues. In some embodiments, a preferred peptide linker typically contains at least 50% glycine residues (e.g., at least 75% glycine residues). In some embodiments, the peptide linker contains only glycine residues. In some embodiments, the peptide linker contains only glycine and serine residues. In some embodiments, such linkers are primarily composed of the amino acids glycine and serine, which are referred to herein as GS-linkers. In some embodiments, the linker contains (GGS)n, where n is 1 to 5 (e.g., 1 to 3) in the sequence. In certain embodiments, the linker contains the sequence (GGGGS)n (SEQ ID NO: 259), where n is 1 to 5 (e.g., 1 to 3) in the sequence. The linker may contain any combination of the above (e.g., two, three, four, or five GS, GGS, and / or GGGGS repeats), and the linkers may be combined.In some embodiments, such linkers have an amino acid length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the linkers are (in single-letter amino acid notation) GGS or GGGGS (SEQ ID NO: 11), GGGGSGGGGS (SEQ ID NO: 9), and GGGGSGGGGSGGGGGS (SEQ ID NO: 23) or GGGGSGGGGSGGGGSGGGGSGGGGGSGGGGGS (SEQ ID NO: 24).

[0283] In some embodiments, the linker sequence between a first antigen-binding domain (e.g., an APP-binding domain such as wild-type SIRPα or a variant thereof) and a second antigen-binding domain (e.g., a shielding domain such as an anti-SIRPα VHH domain) includes a GS linker at the N-terminus and C-terminus of a protease-cleavable linker, i.e., a linker sequence having the sequence GS linker-protease-cleavable linker-GS linker. In some embodiments, the linker includes GGGGS (SEQ ID NO: 11), a protease-cleavable linker, and a sequence in which GGGGS (SEQ ID NO: 11) is arranged from the N-terminus to the C-terminus. In some embodiments, the linker sequence includes GGGGSGGGGS (SEQ ID NO: 9), a protease-cleavable linker, and a sequence in which GGGGSGGGGS (SEQ ID NO: 9) is arranged from the N-terminus to the C-terminus. In some embodiments, the linker sequence includes GGGGS (SEQ ID NO: 11), a protease-cleavable linker, and a sequence in which GGGSGGGGS (SEQ ID NO: 9) are arranged from the N-terminus to the C-terminus.

[0284] In some embodiments, the total length of the linker sequence between the first antigen-binding domain (e.g., an APP-binding domain such as wild-type SIRPα or a variant thereof) and the second antigen-binding domain (e.g., a shielding domain such as an anti-SIRPα VHH domain) is 50 amino acids or less. In some embodiments, the linker is 10-50 amino acids long, 10-40 amino acids long, 10-30 amino acids long, 10-20 amino acids long, 20-50 amino acids long, 20-40 amino acids long, 20-30 amino acids long, 30-50 amino acids long, 30-40 amino acids long, or 40-50 amino acids long.

[0285] D. Target cell antigens and target cell binding domains The conjugates encompassed by this disclosure may comprise at least one binding domain that binds to a target cell antigen (e.g., a cancer antigen) and at least one binding domain that binds to APP. In some embodiments, the target cell antigen is expressed on cells targeted for myeloid cell activity (e.g., direct and / or indirect death). In some embodiments, the conjugates encompassed by this disclosure comprise one target cell binding domain that recognizes a target cell antigen expressed on cells targeted for regulation (e.g., direct and / or indirect death). In some embodiments, the conjugates encompassed by this disclosure comprise two or more target cell binding domains (e.g., two, three, four, or more) that recognize a target cell antigen expressed on cells targeted for regulation (e.g., direct and / or indirect death). In some embodiments, the conjugates encompassed by this disclosure comprise two target cell binding domains, each target cell binding domain recognizing a different target cell antigen expressed on cells targeted for regulation (e.g., direct and / or indirect death). In some embodiments, the binding agents encompassed by this disclosure comprise two or more target cell binding domains (e.g., two, three, four, or more) that collectively recognize at least two target cell antigens expressed on cells targeted for regulation (e.g., direct and / or indirect death). Various target cell antigens and target cell binding domains are known in the art. In some embodiments, the target cell antigen is a microbial antigen. In some embodiments, the target cell antigen is a peptide-major histocompatibility complex (pMHC). In some embodiments, the target cell antigen is a tumor-associated antigen (TAA). In some embodiments, the target cell antigen is a tumor-specific antigen (TSA). In some embodiments, the solid tumor antigen (e.g., TAA or TSA) is expressed by cancer cells from a solid tumor.

[0286] In some embodiments, the cancer antigen (e.g., TAA) is selected from the group of TAAs listed in the table below, or derived from the targets listed in the table below.

[0287] [Table 2] TIFF2026529669000009.tif204165TIFF2026529669000010.tif220165TIFF2026529669000011.tif202165TIFF2026529669000012.tif214165TIFF2026529669000013.tif202165TIFF2026529669000014.tif204165TIFF2026529669000015.tif226165TIFF2026529669000016.tif167165TIFF2026529669000017.tif95165

[0288] Representative antigen-binding domains are well known in the art (see, for example, U.S. Patent No. 11,459,394), and are not limited to such antigen-binding domains, but include, 3F8 targeting GD2 ganglioside, cantuzumab targeting MUC1, 8H9 targeting B7-H3, eclomeximab targeting GD3 ganglisode, nimotuzumab targeting EGFR, necitumumab targeting EGFR, cetuximab targeting EGFR, P2X targeting EGFR, 11F6 targeting EGFR, and other anti-EGFR antibodies (erlotinib, osimertinib, neratinib, gefitinib, panitum targeting EGFR). Mab, dacomitinib, lapatinib, mobocertinib, and vandetanib, etc.), figtumumab targeting IGF1R, cerivanthumab targeting ERBB3, fanvotumab targeting TYRP1, lmab362 targeting Cldn18.2, durigotuzumab targeting HER3, adekatumumab targeting EPCAM, trastuzumab targeting HER2, gilentuximab targeting CA9, nesitumumab targeting EGFR, tucotzumab targeting EPCAM, and zatuximab targeting HER1, 5B1 targeting Ca19-9 (e.g., PCT Publication No. WO2015053871, Sawada) (See et al. Clin Cancer Res. 2011 Mar 1;17(5):1024-1032), it contains antigen-binding domains obtained from r7E3 (Sawada et al.) which targets Ca19-9, or 121SLE (Sawada et al.) which targets Ca19.9.

[0289] In embodiments of the multispecific antigen-binding constructs provided herein, the third antigen-binding domain is a tumor-targeting domain. In some embodiments of the multispecific antigen-binding constructs provided herein, the third antigen-binding domain is an anti-EGFR antibody or its binding fragment.

[0290] In some embodiments, the multispecific binding construct targets GD2 ganglioside (3F8), MUC1 (cantuzumab), B7-H3 (8H9), GD3 ganglisode (ecromeximab), EGFR (nimotuzumab), EGFR (necitumumab), EGFR (cetuximab), EGFR (P2X), EGFR (11F6), IGF1R (figitumumab), and ERBB3. This includes antibodies or antigen-binding fragments selected from cerivanthumab, fanvotumab targeting TYRP1, lmab362 targeting Cldn18.2, durigotuzumab targeting HER3, adekatumumab targeting EPCAM, trastuzumab targeting HER2, girentuximab targeting CA9, nesitumumab targeting EGFR, tucotzumab targeting EPCAM, zatuximab targeting HER, and 5B1 targeting Ca19.9.

[0291] In some embodiments, the multispecific conjugation construct comprises an anti-EGFR antibody or its conjugation fragment. In some embodiments, the antigen-binding domain (e.g., a third antigen-binding domain) binds to EGFR. In some embodiments, the antigen-binding domain is derived from an antibody selected from the group consisting of necitumumab (11F8), cetuximab, nimotuzumab, and P2X. In some embodiments, the multispecific conjugation construct comprises an anti-EGFR antigen-binding fragment that is a Fab. In some embodiments, the multispecific conjugation construct comprises an anti-EGFR antigen-binding fragment that is a single-stranded variable fragment (scFv). In some embodiments, the multispecific conjugation construct comprises an scFv linked to an Fc. In some embodiments, the multispecific conjugation construct comprises a Fab linked to an Fc. In some embodiments, the antigen-binding domain is a Fab.

[0292] In some embodiments, the Fab is a necitumumab Fab comprising a heavy chain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and a light chain comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2. In some embodiments, the Fab comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the Fab is a necitumumab Fab comprising a heavy chain c...

Claims

1. (i) A first antigen-binding domain that binds to and inhibits antiphagocytic proteins (APPs), (ii) A second antigen-binding domain that binds to the first antigen-binding domain and inhibits or reduces the interaction between the first antigen-binding domain and the APP, (iii) A linker containing a linker that can be cleaved in a proteolytic manner, (iv) A third antigen-binding domain that binds to the first target cell antigen, (v) Immunoglobulin Fc region and A multispecific antigen-binding construct comprising, The linker, which includes the protein-degradable linker, links the second antigen-binding domain to the first antigen-binding domain or the immunoglobulin Fc region. The multispecific antigen-binding construct.

2. The multispecific antigen-binding construct according to claim 1, wherein the first antigen-binding domain and the second antigen-binding domain are linked by a linker containing the proteolytically cleavable linker.

3. The multispecific antigen-binding construct according to claim 1, wherein the immunoglobulin Fc region and the second antigen-binding domain are linked by a linker containing the proteolytically cleavable linker.

4. When the linker is in an uncleaved state, the second antigen-binding domain inhibits or reduces the binding of the first antigen-binding domain to the APP, If the linker is cleaved in a proteolytic manner, the second antigen-binding domain does not interfere with the binding of the first antigen-binding domain to the APP. A multispecific antigen-binding construct according to any one of claims 1 to 3.

5. The multispecific antigen-binding construct according to any one of claims 1 to 4, wherein the APP is selected from the group consisting of differentiation antigen group 47 (CD47), differentiation antigen group 24 (CD24), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), β2-microglobulin (B2M), major histocompatibility complex class I (MHC-I), programmed cell death 1 (PD-1), signal regulatory protein α (SIRPα), sialic acid-binding immunoglobulin-like lectin 10 (SIGLEC10), leukocyte immunoglobulin-like receptor 1 (LILRB1), and leukocyte immunoglobulin-like receptor 2 (LILRB2).

6. The first antigen-binding domain described above is (1) Inhibit the interaction between the APP and its binding partner on myeloid cells, or (2) Inhibit the interaction between the APP and its binding partner on cells targeted for myeloid cell activity, A multispecific antigen-binding construct according to any one of claims 1 to 5.

7. The multispecific antigen-binding construct according to claim 6, wherein the myeloid cells are macrophages, dendritic cells, monocytes, neutrophils, tumor-associated macrophages (TAMs), tumor-infiltrating macrophages (TIMs), or myeloid-derived immunosuppressive cells (MDSCs).

8. The binding of the first antigen-binding domain to the APP and its inhibition are Interactions between CD47 and SIRPα, interaction between CD24 and SIGLEC10, interaction between PD-1 and PD-1 ligand (PD-L1 or PD-L2), interaction between LILRB1 ligand (β2M or MHC-I complex) and LILRB1, and interaction between LILRB2 ligand and LILRB2 It inhibits interactions selected from the group consisting of, Optionally, the first antigen-binding domain is: SIRPα or its domain or fragment, SIGLEC10 or its domain or fragment, PD-1 or its domain or fragment, LILRB1 or its domain or fragment, LILRB2 or its domain or fragment, PD-L1 or its domain or fragment, PD-L2 or its domain or fragment, CD47 or its domain or protein, CD24 or its domain or protein, β2M or its domain or protein, or proteins of the MHC-I complex (HLA-A, HLA-B, or HLA-C) That is, A multispecific antigen-binding construct according to any one of claims 1 to 7.

9. The multispecific antigen-binding construct according to any one of claims 1 to 8, wherein the APP is CD47.

10. The multispecific antigen-binding construct according to claim 9, wherein the first antigen-binding domain binds to CD47 and inhibits the interaction between CD47 and wild-type SIRPα, and optionally, the first antigen-binding domain binds to wild-type cell surface-expressed CD47 and inhibits the interaction between wild-type cell surface-expressed CD47 and wild-type cell surface-expressed SIRPα.

11. The first antigen-binding domain described above is (a) Extracellular domain (ECD) of cell surface-expressed proteins, (b) The binding fragment of the ECD of the cell surface expressed protein, or (c) Variants of the ECD or binding fragment of the cell surface-expressed protein that have been modified to improve binding to the APP. A multispecific antibody-conjugated construct according to any one of claims 1 to 10, comprising:

12. The cell surface-expressed protein is wild-type SIRPα, and the first antigen-binding domain is (a) ECD of the wild-type SIRPα, (b) The binding fragment of wild-type SIRPα, or (c) Variants of the ECD or binding fragment of the wild-type SIRPα that have been manipulated to improve binding to the APP. This includes, where APP is CD47, The multispecific antibody-binding construct according to claim 11.

13. The multispecific antibody-binding construct according to claim 11 or claim 12, wherein the binding fragment comprises, optionally, the immunoglobulin-variable (V) region (domain 1) of the ECD of the cell surface-expressed protein, or of the ECD of the wild-type SIRPα.

14. The first antigen-binding domain described above is The domain of wild-type SIRPα that binds to CD47, or A variant comprising one or more amino acid substitutions in the domain of the wild-type SIRPα that improves binding to CD47. A multispecific antibody-conjugated construct according to any one of claims 1 to 10, comprising:

15. (i) (a) The domain of wild-type SIRPα that binds to antiphagocytic proteins (APP), or (b) A variant comprising one or more amino acid substitutions in the domain of the wild-type SIRPα that improves binding to the APP. A first antigen-binding domain comprising, wherein the APP is CD47, A second antigen-binding domain which is an anti-SIRPα antibody or antigen-binding fragment that binds to the first antigen-binding domain and inhibits or reduces the interaction between the first antigen-binding domain and the APP, and A multispecific antigen-binding construct comprising, The first antigen-binding domain and the second antigen-binding domain are linked by a linker containing a linker that can be cleaved in a proteolytic manner. The multispecific antigen-binding construct.

16. The multispecific antigen-binding construct according to any one of claims 1 to 15, wherein the first antigen-binding domain has an amino acid length of 100 to 120, optionally 106 to 118, and more optionally 112 to 118.

17. The multispecific antigen-binding construct according to any one of claims 15 to 16, wherein the domain of wild-type SIRPα is the extracellular domain of SIRPα.

18. The multispecific antigen-binding construct according to any one of claims 14 to 16, wherein the domain of wild-type SIRPα is an immunoglobulin variable region (IgV).

19. The multispecific antigen-binding construct according to any one of claims 12 to 18, wherein the wild-type SIRPα is wild-type human SIRPα.

20. The multispecific antigen-binding construct according to any one of claims 1 to 19, wherein the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

103.

21. The multispecific antigen-binding construct according to any one of claims 1 to 19, wherein the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

103.

22. The multispecific antigen-binding construct according to any one of claims 1 to 21, wherein the first antigen-binding domain is the sequence shown in SEQ ID NO: 103 or comprises the same.

23. The multispecific antigen-binding construct according to any one of claims 1 to 19, wherein the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

104.

24. The multispecific antigen-binding construct according to any one of claims 1 to 19, wherein the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

104.

25. The multispecific antigen-binding construct according to any one of claims 1 to 19, 23, and 24, wherein the first antigen-binding domain is the sequence shown in SEQ ID NO: 104 or comprises the same.

26. The first antigen-binding domain described above is A variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα to improve binding to CD47. The multispecific antigen-binding construct according to any one of claims 1 to 21, 23, and 24.

27. The variant SIRPα has a dissociation constant (K) of less than 100 nanomolar (nM), less than 10 nM, less than 1 nM, less than 100 picomolar (pM), less than 10 pM, or less than 1 pM, or any combination of the above. Ｄ The multispecific antigen-binding construct according to claim 26, which binds to wild-type human CD47.

28. The variant SIRPα has a dissociation constant (K) of less than 100 nanomolar (nM) and is arbitrarily selected from 1 nM to 100 nM, 1 nM to 75 nM, 1 nM to 50 nM, 1 nM to 25 nM, 1 nM to 10 nM, 10 nM to 100 nM, 10 nM to 75 nM, 10 nM to 50 nM, 10 nM to 25 nM, 25 nM to 100 nM, 25 nM to 75 nM, 25 nM to 50 nM, or 50 nM to 100 nM, 50 nM to 75 nM, or 75 nM to 100 nM. Ｄ A multispecific antigen-binding construct according to claim 26 or claim 27, which binds to wild-type human CD47.

29. The variant SIRPα is less than 1 nM, and arbitrarily selected, has a dissociation constant (K) of 100 pM to 1 nM, 100 pM to 750 pM, 100 pM to 500 pM, 100 pM to 250 pM, 250 pM to 1 nM, 250 pM to 750 pM, 250 pM to 500 pM, 500 pM to 1 nM, 500 pM to 750 pM, or 750 pM to 1 nM. Ｄ A multispecific antigen-binding construct according to claim 26 or claim 27, which binds to wild-type human CD47.

30. The aforementioned variant SIRPα has a dissociation constant (K) of less than 100 picomolar concentration (pM). Ｄ A multispecific antigen-binding construct according to claim 26 or claim 27, which binds to wild-type human CD47.

31. The variant SIRPα has a dissociation constant (K) of 1 pM to 100 pM, and optionally 1 pM to 75 pM, 1 pM to 50 pM, 1 pM to 25 pM, 1 pM to 10 pM, 10 pM to 100 pM, 10 pM to 75 pM, 10 pM to 50 pM, 10 pM to 25 pM, 25 pM to 100 pM, 25 pM to 75 pM, 25 pM to 50 pM, or 50 pM to 100 pM, 50 pM to 75 pM, or 75 pM to 100 pM. Ｄ A multispecific antigen-binding construct according to claim 26, claim 27, or claim 30, which binds to wild-type human CD47.

32. The one or more amino acid substitutions are: L4F or L4I or L4V, V6F or V6I or V6L, V27F or V27I or V27L (A27F or A27I or A27L), I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L or H56R, S66G or S66T or S6 A multispecific antigen-binding construct according to any one of claims 14, 15, and 26-31, selected from the group consisting of 6A (or L66G or L66T or L66A), K68R, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

33. A multispecific antigen-binding construct according to any one of claims 14, 15, and 26-32, wherein at least one amino acid substitution is E54Q (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

34. The multispecific antigen-binding construct according to any one of claims 14, 15, and 26-32, wherein the one or more amino acid substitutions include K53R, E54Q, and S66T (L66T) (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

35. The one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I; or V6I, V27I (or A27I), I31F, E47L, K53R, E54Q, H56P, S66T (or L66T); or L4V, V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56 A multispecific antigen-binding construct according to any one of claims 14, 15, and 26-34, wherein the construct is P, V63I, S66T (or L66T), K68R, V92I; or V6I, V27I (or A27I), I31T, E47V, K53R, E54Q, H56P, S66G (or L66G), K68R, V92I, F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

36. The multispecific antigen-binding construct according to any one of claims 14, 15, and 26-35, wherein the one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

37. The multispecific antigen-binding construct according to any one of claims 1 to 21, 23, 24, and 26 to 36, wherein the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

105.

38. The multispecific antigen-binding construct according to any one of claims 1 to 21, 23, 24, and 26 to 36, wherein the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

105.

39. The multispecific antigen-binding construct according to any one of claims 1 to 21, 23, 24, and 26 to 38, wherein the first antigen-binding domain is the amino acid sequence shown in SEQ ID NO: 105 or comprises the same.

40. The multispecific antigen-binding construct according to any one of claims 1 to 39, wherein the first antigen-binding domain is deglycosylated.

41. The multispecific antigen-binding construct according to any one of claims 14, 15, and 26-40, wherein at least one of the one or more amino acid substitutions is N80A (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

42. The multispecific antigen-binding construct according to any one of claims 14, 15, 26-36, 40, and 41, wherein the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

10.

43. The multispecific antigen-binding construct according to any one of claims 14, 15, 26-36, 40, and 41, wherein the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

10.

44. The multispecific antigen-binding construct according to any one of claims 14, 15, 26-36, and 40-43, wherein the first antigen-binding domain is the amino acid sequence shown in SEQ ID NO: 10 or comprises the same.

45. The multispecific antigen-binding construct according to any one of claims 14, 15, 26-36, 40, and 41, wherein the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

27.

46. The multispecific antigen-binding construct according to any one of claims 14, 15, 26-36, 40, and 41, wherein the first antigen-binding domain is represented by an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

27.

47. The multispecific antigen-binding construct according to any one of claims 14, 15, 26-36, 40, 41, 45, and 46, wherein the first antigen-binding domain is the amino acid sequence shown in SEQ ID NO: 27 or comprises the same.

48. The multispecific antigen-binding construct according to any one of claims 1 to 10, wherein the first antigen-binding domain is an anti-CD47 antibody or an antigen-binding fragment that binds to CD47.

49. The first antigen-binding domain is a single-domain antibody, a single-strand variable fragment (scFv), sc(Fv) ２ , Fab, Fv, Fav, F(ab') ２ The multispecific antigen-binding construct according to claim 48, wherein Fab', dsFv, Fde, and sdFv.

50. The multispecific antigen-binding construct according to claim 48 or claim 49, wherein the first antigen-binding domain is a single-domain antibody that is VHH.

51. The multispecific antigen-binding construct according to claim 50, wherein the VHH is a camel heavy chain antibody, a humanized VHH domain, an affinity-mature VHH domain, or a human VHH domain.

52. The multispecific antigen-binding construct according to any one of claims 1 to 10 and 48 to 51, wherein the first antigen-binding domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

208.

53. The multispecific antigen construct according to any one of claims 1 to 10 and 48 to 52, wherein the first antigen-binding portion includes the sequence shown in Sequence ID No.

208.

54. The second antigen-binding domain described above is A dissociation constant for binding to the first antigen-binding domain that is greater than the dissociation constant for the first antigen-binding domain to the APP. A multispecific antigen-binding construct according to any one of claims 1 to 53, having the above characteristics.

55. The multispecific antigen-binding construct according to claim 54, wherein the dissociation constant (Kd) of the second antigen-binding domain relative to the first antigen-binding domain is at least 2, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times greater than the dissociation constant of the first antigen-binding domain relative to the APP.

56. The multispecific antigen-binding construct according to any one of claims 1 to 55, wherein when the cleavable linker is cleaved, the second antigen-binding domain does not interfere with or compete with the first antigen-binding domain for binding to the APP.

57. The second antigen-binding domain described above is Dissociation constant for binding to the first antigen-binding domain, which is 1 nM or greater, and optionally 100 nM to 1 μM, 10 nM to 1 μM, or 1 nM to 1 μM. A multispecific antigen-binding construct according to any one of claims 1 to 56, having the above characteristics.

58. The second antigen-binding domain described above is Dissociation constant for binding to the first antigen-binding domain, which is 1 nM or greater. A multispecific antigen-binding construct according to any one of claims 1 to 57, having the above characteristics.

59. The second antigen-binding domain described above is Dissociation constant for binding to the first antigen-binding domain, which is 10 nM or greater. A multispecific antigen-binding construct according to any one of claims 1 to 58, having the above characteristics.

60. The second antigen-binding domain described above is Dissociation constant for binding to the first antigen-binding domain, which is 100 nM or greater. A multispecific antigen-binding construct according to any one of claims 1 to 58, having the above characteristics.

61. The second antigen-binding domain described above is Dissociation constant for binding to the first antigen-binding domain, which is 1 μM or larger. A multispecific antigen-binding construct according to any one of claims 1 to 58, having the above characteristics.

62. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 61, wherein the second antigen-binding domain is an antibody or an antigen-binding fragment.

63. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 62, wherein the second antigen-binding domain is an anti-SIRPα antibody or an antigen-binding fragment.

64. The antibody or antigen-binding fragment is a single domain antibody, a single-chain variable fragment (scFv), sc(Fv) ２ , Fab, Fv, Fav, F(ab') ２ , Fab', dsFv, Fde, or sdFv, The multispecific antigen-binding construct according to claim 15, claim 62, or claim 63.

65. The multispecific antigen-binding construct according to any one of claims 1 to 64, wherein the second antigen-binding domain is a single-domain antibody that is VHH.

66. The multispecific antigen-binding construct according to claim 65, wherein the VHH is a camel heavy chain antibody, a humanized VHH domain, an affinity-mature VHH domain, or a human VHH domain.

67. The multispecific antigen-binding construct according to claim 65 or claim 66, wherein the VHH comprises a complementarity-determining region 1 (CDR1) containing an amino acid sequence selected from SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, and 45; a complementarity-determining region 2 (CDR2) containing an amino acid sequence selected from SEQ ID NOs: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and a complementarity-determining region 3 (CDR3) containing an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.

68. The SIRPα VHH domain corresponds to SEQ ID NOs. 37, 46, and 61, SEQ ID NOs. 38, 46, and 61, SEQ ID NOs. 39, 47, and 62, SEQ ID NOs. 40, 48, and 63, SEQ ID NOs. 41, 49, and 64, SEQ ID NOs. 37, 50, and 61, SEQ ID NOs. 42, 51, and 65, SEQ ID NOs. 43, 52, and 66, SEQ ID NOs. 37, 53, and 67, SEQ ID NOs. 44, 54, and 68, and SEQ ID NOs. A multispecific antigen-binding construct according to any one of claims 65 to 67, comprising CDR1, CDR2, and CDR3 shown in 43, 55, and 63, SEQ ID NOs: 40, 56, and 69, respectively; SEQ ID NOs: 37, 57, and 70, respectively; SEQ ID NOs: 40, 55, and 63, respectively; SEQ ID NOs: 41, 58, and 71, respectively; SEQ ID NOs: 43, 59, and 72, respectively; SEQ ID NOs: 37, 60, and 73, respectively; or SEQ ID NOs: 45, 56, and 73, respectively.

69. The aforementioned VHH domain, The amino acid sequence is one of the sequences shown in any one of SEQ ID NOs: 13-21 and 28-36, or the amino acid sequence is one of the sequences shown in any one of SEQ ID NOs: 13-21 and 28-36 and exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and Binds to SIRPα, A multispecific antigen-binding construct according to any one of claims 65 to 68.

70. The multispecific antigen-binding construct according to any one of claims 65 to 68, wherein the VHH domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 13 to 21 and 28 to 36.

71. The multispecific antigen-binding construct according to any one of claims 65 to 70, wherein the VHH domain binds to the IgV domain of wild-type human SIRPα or a variant thereof.

72. The anti-SIRPα antibody or antigen-binding fragment is panreactive, Wild-type SIRPα, and At least one variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα to improve binding to CD47. A multispecific antigen-binding construct according to any one of claims 15 and 63 to 71, which binds to a.

73. The aforementioned at least one variant SIRPα is One or more amino acid substitutions in the IgV domain of the wild-type SIRPα to improve binding to CD47 A multispecific antigen-binding construct according to claim 71 or claim 72, comprising:

74. The IgV domain or a variant of the wild-type human SIRPα (i) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103, (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 104, (iii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 105, (iv) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10, and (v) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27 A multispecific antigen-binding construct according to any one of claims 71 to 73, selected from among them.

75. The anti-SIRPα antibody or antigen-binding fragment, (1) IgV domain of wild-type allele SIRPα, optionally, IgV domain of wild-type allele 1 and / or wild-type allele 2 SIRPα, and (2) (a) At least one IgV domain of a variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47. A multispecific antigen-binding construct according to any one of claims 71 to 74, which binds to a.

76. The anti-SIRPα antibody or antigen-binding fragment binds to wild-type human SIRPα, and optionally binds to the IgV domain of the wild-type human SIRPα, and the wild-type human SIRPα (i) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103, or an optional wild-type human SIRPα containing the amino acid sequence shown in SEQ ID NO: 103, or (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 104, or optionally, wild-type human SIRPα containing the amino acid sequence shown in SEQ ID NO:

104. A multispecific antigen-binding construct according to any one of claims 71 to 75, comprising:

77. The anti-SIRPα antibody or antigen-binding fragment binds to variant SIRPα, and optionally binds to the IgV domain of variant SIRPα, where the variant SIRPα L4F or L4I or L4V, V6F or V6I or V6L, V27F or V27I or V27L (A27F or A27I or A27L), I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L or H56R, S66G or S66T or S66A (or L66G or L66T or L66A), K68R, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104) A multispecific antigen-binding construct according to any one of claims 71 to 75, comprising one or more amino acid substitutions in wild-type SIRPα, selected from the group consisting of the following.

78. The multispecific antigen-binding construct according to claim 77, wherein the one or more amino acid substitutions include K53R, E54Q, and S66T (L66T) (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

79. The one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I; or V6I, V27I (or A27I), I31F, E47L, K53R, E54Q, H56P, S66T (or L66T); or L4V, V6I, V27I (or A27I), I31F, E47V, K53R, E54 A multispecific antigen-binding construct according to claim 77 or claim 78, wherein the construct is Q, H56P, V63I, S66T (or L66T), K68R, V92I; or V6I, V27I (or A27I), I31T, E47V, K53R, E54Q, H56P, S66G (or L66G), K68R, V92I, F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

80. The multispecific antigen-binding construct according to any one of claims 77 to 79, wherein the one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

81. The multispecific antigen-binding construct according to any one of claims 71 to 80, wherein the variant SIRPα is named FB3, FD6, FA4, or CV1.

82. The anti-SIRPα antibody or antigen-binding fragment binds to variant SIRPα, and optionally binds to the IgV domain of variant SIRPα, and the variant SIRPα or the IgV domain of variant SIRPα (iii) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 105, (iv) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10, (v) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27 A multispecific antigen-binding construct according to any one of claims 71 to 81, comprising:

83. The dissociation constant (K) of wild-type human SIRPα or its variant relative to wild-type human CD47. Ｄ A K that is at least 2 times, 5 times, 10 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times larger than ) Ｄ The multispecific antigen-binding construct according to any one of claims 71 to 82, wherein the anti-SIRPα antibody or antigen-binding fragment binds to the wild-type human SIRPα or its variant, and optionally, the CD47 is cell surface-expressed CD47.

84. The multispecific antigen-binding construct according to any one of claims 1 to 83, wherein the linker is a substrate of a protease, optionally the protease is an extracellular protease, and / or the linker is a substrate of renin, pepsin C, napsin A, matrix metalloproteinase (MMP), matryptase, urokinase-type plasminogen activator (uPA), disintegrin and metalloproteinase (ADAM), disintegrin and metalloproteinase having a thrombospondin motif (ADAMTS), regmine, urokinase, or hepsin.

85. The multispecific antigen-binding construct according to any one of claims 1 to 84, wherein the linker that can be cleaved in a proteolytic manner is a polypeptide that functions as a substrate for a protease.

86. The multispecific antigen-binding construct according to claim 85, wherein the protease is produced by a tumor or by cells present in the tumor microenvironment.

87. The multispecific antigen-binding construct according to claim 85 or claim 86, wherein the protease is selected from matryptase, matrix metalloproteinase (MMP), granzyme B, and combinations thereof.

88. The multispecific antigen-binding construct according to any one of claims 85 to 87, wherein the protease is a matryptase.

89. The multispecific antigen-binding construct according to any one of claims 1 to 88, wherein the linker capable of proteolytic cleavage is VHMPPLGFLGPRQARVVN (SEQ ID NO: 22).

90. The multispecific antigen-binding construct according to any one of claims 1 to 89, wherein the linker, which includes the proteolytically cleavable linker, includes an N-terminal and / or C-terminal GS linker sequence.

91. The multispecific antigen-binding construct according to claim 90, wherein the GS linker sequence is the sequence (GGGGGS)n (where n is 1 to 5) (SEQ ID NO: 259), and optionally the GS linker sequence is GGGGSGGGGGS (SEQ ID NO: 9) or GGGGS (SEQ ID NO: 11).

92. The multispecific antigen-binding construct according to any one of claims 15 to 47 and 54 to 91, wherein the construct further comprises a third antigen-binding domain that binds to a first target cell antigen.

93. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 92, wherein the first target cell antigen is expressed on cells targeted for myeloid cell activity.

94. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 93, wherein the third antigen-binding domain is an antibody or an antigen-binding fragment.

95. The antibody or the antigen-binding fragment is a single-stranded variable fragment (scFv), sc(Fv) ２ , Fab, Fv, Fav, F(ab') ２ The multispecific antigen-binding construct according to claim 94, which is Fab', dsFv, Fde, sdFv, or a single-domain antibody (sdAb).

96. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 95, wherein the first target cell antigen is a microbial antigen, a peptide-major histocompatibility complex (pMHC), or a tumor-associated antigen (TAA).

97. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 96, wherein the first target cell antigen is a TAA, and the TAA is selected from the group of TAAs listed in Table 2 or derived from the targets listed in Table 2.

98. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 97, wherein the third antigen-binding domain is Fab.

99. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 97, wherein the third antigen-binding domain is a single-chain antibody fragment.

100. The multispecific antigen-binding construct according to any one of claims 1 to 14, 16 to 97, and 99, wherein the third antigen-binding domain is VHH.

101. The multispecific antigen-binding construct according to claim 100, wherein the VHH is a camel heavy chain antibody, a humanized VHH domain, an affinity-mature VHH domain, or a human VHH domain.

102. The multispecific antigen-binding construct according to any one of claims 1 to 14, 16 to 97, 100, and 101, wherein the third antigen-binding domain is a single-stranded variable fragment (scFv).

103. The multispecific antigen-binding construct according to any one of claims 99 to 102, wherein the third antigen-binding domain comprises two different single-chain antibody fragments.

104. The multispecific antigen-binding construct according to claim 103, wherein the third antigen-binding domain is a biparatopic.

105. It further comprises a fourth antigen-binding domain that binds to a second target cell antigen, Optionally, the second target cell antigen is expressed on the cells targeted for myeloid cell activity. A multispecific antigen-binding construct according to any one of claims 1 to 104.

106. The multispecific antigen-binding construct according to claim 105, wherein the fourth antigen-binding domain is an antibody or an antigen-binding fragment.

107. The antibody or the antigen-binding fragment is a single-stranded variable fragment (scFv), sc(Fv) ２ , Fab, Fv, Fav, F(ab') ２ The multispecific antigen-binding construct according to claim 106, which is Fab', dsFv, Fde, sdFv, or a single-domain antibody (sdAb).

108. The multispecific antigen-binding construct according to any one of claims 105 to 107, wherein the fourth antigen-binding domain is Fab.

109. The multispecific antigen-binding construct according to any one of claims 103 to 108, wherein the second target cell antigen is a microbial antigen, a peptide-major histocompatibility complex (pMHC), or a tumor-associated antigen (TAA).

110. The multispecific antigen-binding construct according to any one of claims 103 to 109, wherein the second target cell antigen is a TAA, and the TAA is selected from the group of TAAs listed in Table 2 or derived from the targets listed in Table 2.

111. A multispecific antigen-binding construct according to any one of claims 15 to 110, further comprising an immunoglobulin Fc region.

112. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 111, wherein the immunoglobulin Fc region is a homodimeric Fc region.

113. The multispecific antigen-binding construct according to claim 112, wherein the third antigen-binding domain is divalent.

114. The multispecific antigen-binding construct according to claim 112 or claim 113, wherein the first antigen-binding domain is divalent.

115. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 114, wherein the immunoglobulin Fc region is a wild-type human IgG1 Fc region.

116. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 115, wherein the immunoglobulin Fc region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

98.

117. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 116, wherein the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO:

98.

118. The multispecific antigen-binding construct according to claim 117, wherein the Fc region is a variant Fc region comprising one or more amino acid mutations or substitutions that increase the antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) activity of the multispecific antigen-binding construct.

119. The multispecific antigen-binding construct according to claim 118, wherein the variant Fc region contains one or more amino acid mutations compared to the wild-type human IgG1 Fc region.

120. The variant Fc region is 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 251, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 272, 279, 280, 281, 282, 283, 284, 292, 293, 295, 296, 297, 298, 299, 300, 304, 305, 309, 313, 316, 31 The molecule contains one or more amino acid mutations at positions selected from the group consisting of 8, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 339, 341, 343, 370, 373, 378, 392, 396, 416, 419, 421, 440, and 443, and optionally, one or more of the above amino acid mutations are one or more amino acid substitutions, and optionally, the above one or more amino acid substitutions are 220S, 229S, 232G, 233P, 234A, 234D, 234E , 234F, 234G, 234H, 234L, 234N, 234Q, 234T, 234V, 234Y, 235A, 235D, 23 5E, 235F, 235G, 235H, 235N, 235P, 235Q, 235R, 235S, 235T, 235W, 235Y, 2 36A, 236E, 236I, 236N, 236P, 236R, 237A, 237K, 237L, 237N, 237P, 238K, 238S, 239D, 239E, 239F, 239H, 239N, 239Q, 239R, 239T, 239Y, 240M, 240T , 241A, 241E, 241L, 241W, 241Y, 243L, 243Q, 243R, 243W, 243Y, 244H, 24 5A, 247G, 247I, 247L, 247V, 24IR, 252Y, 254T, 255L, 256E, 256M, 25IF, 2 62E, 262T, 263M, 263T, 264A, 264E, 264F, 264L, 264M, 264R, 264T, 264Y, 265A, 265F, 265G, 265H, 265N, 265Q, 265T, 265V, 265Y, 266M, 266T, 267E267L, 267Q, 267R, 268E, 268Q, 269F, 269G, 269H, 269R, 269Y, 270E, 270H, 280A, 284M, 292L, 292P, 296D, 2 96E, 296L, 296N, 296Q, 296S, 296T, 297A, 297D, 297E, 297S, 298A, 298F, 298H, 299A, 299E, 299F, 299H, 29 9I, 299S, 299V, 300L, 305I, 309L, 316D, 318A, 324T, 325A, 325E, 325H, 325L, 325Q, 325T, 325V, 326W, 327 G, 327L, 327N, 327R, 327W, 328A, 328D, 328E, 328F, 328H, 328M, 328N, 328Q, 328R, 328S, 328T, 329F, 329H, 329K, 329Q, 330C, 330F, 330G, 330H, 330I, 330K, 330L, 330N, 330P, 330R, 330S, 330T, 330V, 330Y, 331A, 3 31D, 331E, 331F, 331G, 331H, 331K, 331L, 331M, 331N, 331Q, 331R, 331S, 331T, 331V, 331W, 331Y, 332A, 33 A multispecific antigen-binding construct according to claim 118 or claim 119, selected from the group consisting of 2D, 332E, 332F, 332H, 332N, 332Q, 332S, 332T, 332W, 332Y, 333A, 333S, 334A, 339Q, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y, and 443W.

121. The multispecific antigen-binding construct according to claim 119 or claim 120, wherein the one or more amino acid mutations are amino acid substitutions G236A, S239D, and I332E.

122. The multispecific antigen-binding construct according to claim 121, wherein the immunoglobulin Fc region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

97.

123. The multispecific antigen-binding construct according to claim 121 or claim 122, wherein the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO:

97.

124. The third antigen-binding region is Fab, and the multispecific antigen-binding construct is A first polypeptide chain comprising the heavy chain variable region (VH) and heavy chain constant region 1 (CH1) of Fab, the immunoglobulin Fc region, the first antigen-binding domain, the linker including the proteolytically cleavable linker, and the second antigen-binding domain, A second polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of the aforementioned Fab, A multispecific antigen-binding construct according to any one of claims 98 and 111 to 123, comprising:

125. The third antigen-binding region is Fab, and the multispecific antigen-binding construct is A first polypeptide comprising the heavy chain variable region (VH) and heavy chain constant region 1 (CH1) of Fab, the immunoglobulin Fc region, the first antigen-binding domain, the linker including the proteolytically cleavable linker, and the second antigen-binding domain, in order from the N-terminus to the C-terminus, A second polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of the aforementioned Fab, A multispecific antigen-binding construct according to any one of claims 98 and 111 to 124, comprising:

126. The multispecific polypeptide construct comprises two identical first polypeptides and two identical second polypeptides. The two first polypeptides are covalently linked by a disulfide bond, and Each of the second polypeptides is covalently linked to one of the first polypeptides by a disulfide bond. The multispecific antigen-binding construct according to claim 124 or claim 125.

127. The third antigen-binding region is Fab, and the multispecific antigen-binding construct is A first polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain variable region (VH) and a heavy chain constant region (CH1) of Fab, an immunoglobulin Fc region containing the amino acid sequence shown in SEQ ID NO: 97, a first antigen-binding domain containing the amino acid sequence shown in SEQ ID NO: 10, a linker containing a proteolytic cleavage linker shown in SEQ ID NO: 12, and a second antigen-binding domain containing a sequence having at least 95% sequence identity with any one of the sequences shown in SEQ ID NOs: 13-21 and 28-36; A second polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of the aforementioned Fab, A multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 126, comprising:

128. The third antigen-binding region is Fab, and the multispecific antigen-binding construct is A first polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain variable region (VH) and a heavy chain constant region (CH1) of Fab, an immunoglobulin Fc region containing the amino acid sequence shown in SEQ ID NO: 97, a first antigen-binding domain containing the amino acid sequence shown in SEQ ID NO: 27, a linker containing a proteolytic cleavage linker shown in SEQ ID NO: 12, and a second antigen-binding domain containing a sequence having at least 95% sequence identity with any one of the sequences shown in SEQ ID NOs: 13-21 and 28-36; A second polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of the aforementioned Fab, A multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 126, comprising:

129. The multispecific antigen-binding construct according to claim 128, wherein the second antigen-binding domain includes the sequence shown in any one of SEQ ID NOs: 13-21 and 28-36.

130. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 129, wherein the multispecific antigen-binding construct comprises a peptide linker between the immunoglobulin Fc region and the first antigen-binding domain.

131. The multispecific antigen-binding construct according to claim 130, wherein the peptide linker is a GS linker.

132. The multispecific antigen-binding construct according to claim 131, wherein the GS linker is (GGGGGS)n (wherein n is 1 to 5) (SEQ ID NO: 259).

133. Claim 131 or the multispecific antigen-binding constrictor according to claim 131, wherein the GS linker is GGGGS (SEQ ID NO: 11), GGGGSGGGGGS (SEQ ID NO: 9), GGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 23), or GGGGSGGGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 24).

134. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 133, wherein the first target cell antigen is EGFR.

135. The multispecific antigen-binding construct according to claim 134, wherein the third antigen-binding domain is a Fab derived from an antibody selected from the group consisting of necitumumab (11F8), cetuximab, nimotuzumab, and P2X.

136. The aforementioned Fab is, (a) A heavy chain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7 and a light chain containing at least 95% sequence identity with SEQ ID NO: 2 (b) A heavy chain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 205 and a light chain having at least 95% sequence identity with SEQ ID NO: 2 (c) A heavy chain containing an amino acid sequence having at least 95% sequence identity with amino acids 1-217 of SEQ ID NO: 93, and a light chain containing at least 95% sequence identity with SEQ ID NO:

94. (d) A heavy chain containing a sequence having at least 95% sequence identity with amino acids 1-221 of SEQ ID NO: 211 and a light chain containing at least 95% sequence identity with SEQ ID NO: 96, or (e) A heavy chain containing a sequence having at least 95% sequence identity with amino acids 1-217 of SEQ ID NO: 212 and a light chain containing at least 95% sequence identity with SEQ ID NO:

213. A multispecific antigen-binding construct according to any one of claims 127 to 135, comprising:

137. The aforementioned Fab is, (a) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2 (b) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 205 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2 (c) A heavy chain containing amino acids 1 to 217 of SEQ ID NO: 93 and a light chain containing the amino acid sequence shown in SEQ ID NO: 94 (d) A heavy chain containing amino acids 1 to 221 of SEQ ID NO: 211 and a light chain containing the amino acid sequence shown in SEQ ID NO: 96, or (e) A heavy chain containing amino acids 1-217 of SEQ ID NO: 212 and a light chain containing the amino acid sequence shown in SEQ ID NO:

213. A multispecific antigen-binding construct according to any one of claims 127 to 136, comprising:

138. The multispecific antigen-binding construct according to any one of claims 127 to 137, wherein the Fab is a necitumumab Fab comprising a heavy chain having at least 95% sequence identity with SEQ ID NO: 7 and a light chain having at least 95% sequence identity with SEQ ID NO:

2.

139. The multispecific antigen-binding construct according to any one of claims 127 to 138, wherein the Fab is a necitumumab Fab comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain containing the amino acid sequence shown in SEQ ID NO:

2.

140. The multispecific antigen-binding construct according to any one of claims 127 to 137, wherein the Fab is a necitumumab Fab comprising a heavy chain having at least 95% sequence identity with SEQ ID NO: 205 and a light chain having at least 95% sequence identity with SEQ ID NO:

2.

141. The multispecific antigen-binding construct according to any one of claims 127 to 137 and 140, wherein the Fab is a necitumumab Fab comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 205 and a light chain containing the amino acid sequence shown in SEQ ID NO:

2.

142. The multispecific antigen-binding construct according to any one of claims 1 to 14, 16 to 21, 23, 24, 40 to 44, 48 to 127, and 129 to 141, wherein the multispecific polypeptide construct comprises a first polypeptide chain having at least 95% sequence identity with the sequence shown in any one of SEQ ID NOs: 110 to 120, and a second polypeptide chain having at least 95% sequence identity with the sequence shown in SEQ ID NOs:

2.

143. The multispecific antigen-binding construct according to any one of claims 1 to 14, 16 to 21, 23, 24, 40 to 44, 48 to 127, and 129 to 142, wherein the multispecific polypeptide construct comprises a first polypeptide chain containing the sequence shown in any one of SEQ ID NOs: 110 to 120, and a second polypeptide chain containing the sequence shown in SEQ ID NO:

2.

144. The multispecific polypeptide construct is A first polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in any one of sequence numbers 125 to 137, and A second polypeptide chain containing a sequence having at least 95% sequence identity with the sequence shown in Sequence ID No.

2. A multispecific antigen-binding construct according to any one of claims 1 to 14, 16, 20 to 21, 23, 24, 26 to 38, 40 to 43, 45 to 127, and 129 to 141, comprising:

145. The multispecific polypeptide construct is A first polypeptide chain comprising the sequence shown in any one of sequence numbers 125 to 137, and A second polypeptide chain containing the sequence shown in Sequence ID No. 2 A multispecific antigen-binding construct according to any one of claims 1 to 14, 16, 20 to 21, 23, 24, 26 to 38, 40 to 43, 45 to 127, 129 to 141, and 144, comprising:

146. The third antigen-binding region is a single-chain antibody fragment, and the multispecific antigen-binding construct is A polypeptide comprising the third antigen-binding region, the Fc region, the first antigen-binding domain, the linker including the proteolytically cleavable linker, and the second antigen-binding domain. A multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 97, 99 to 104, or 111 to 123, comprising:

147. A multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 97, 99 to 104, 111 to 123, and 146, wherein the third antigen-binding region is a single-chain antibody fragment, and the multispecific antigen-binding construct comprises the third antigen-binding region, the Fc region, the first antigen-binding domain, the linker including the proteolytically cleavable linker, and the second antigen-binding domain in order from the N-terminus to the C-terminus.

148. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 97, 99 to 104, 111 to 123, 146, and 147, wherein the immunoglobulin Fc region is a variant Fc region comprising a modified hinge domain including the substitution of amino acids EPKSC to EPKSS.

149. The multispecific antigen-binding construct according to claim 148, wherein the immunoglobulin Fc region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

102.

150. The immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO: 102, according to claim 148 or the multispecific antigen-binding construct according to claim 148.

151. The multispecific antigen-binding construct according to any one of claims 1 to 14 and 16 to 97, 99 to 104, 111 to 123, and 146 to 150, wherein the first target cell antigen is EGFR.

152. The multispecific antigen-binding construct according to claim 151, wherein the third antigen-binding domain is an scFv selected from the group consisting of necitumumab (11F8), cetuximab, nimotuzumab, and P2X.

153. The multispecific antigen-binding construct according to claim 151, wherein the third antigen-binding domain is an scFv derived from necitumumab (11F8).

154. The aforementioned scFv is, A variable weight (VH) chain containing an amino acid sequence having at least 95% sequence identity with the VH chain sequence present in Sequence ID No. 207, A variable light (VL) chain containing an amino acid sequence having at least 95% sequence identity with the VL chain sequence present in Sequence ID No. 207, A multispecific antigen-binding construct according to claim 153, comprising:

155. The multispecific antigen-binding construct according to claim 153, wherein the scFv comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

207.

156. The multispecific antigen-binding construct according to any one of claims 153 to 155, wherein the scFv comprises the amino acid sequence shown in SEQ ID NO:

207.

157. The multispecific antigen-binding construct according to any one of claims 1 to 122, wherein the Fc region is a heterodimeric Fc region.

158. The multispecific antigen-binding construct according to any one of claims 1 to 122 and 157, wherein the first antigen-binding domain or the third antigen-binding domain is bivalent, and the remaining one of the first antigen-binding domain and the third antigen-binding domain is monovalent.

159. The multispecific antigen-binding construct according to claim 158, wherein the first antigen-binding domain is bivalent and the third antigen-binding domain is monovalent.

160. The multispecific antigen-binding construct according to claim 158, wherein the first antigen-binding domain is monovalent and the third antigen-binding domain is bivalent.

161. It comprises a first antigen-binding domain that binds to an antiphagocytic protein (APP), a second antigen-binding domain that binds to the first antigen-binding domain, a heterodimer Fc region containing a first Fc polypeptide and a second Fc polypeptide, and a third antigen-binding domain that is a target cell antigen-binding domain that binds to a target cell antigen expressed on cells targeted for myeloid cell activity. The second antigen-binding domain is linked to either the first or second Fc polypeptide by a proteolytically cleavable linker. A multispecific antigen-binding construct according to any one of claims 1 to 122 and 157 to 160.

162. (1) A first heavy chain comprising the first polypeptide chain and the first antigen-binding domain of the heterodimer Fc, (2) The second heavy chain comprising the second polypeptide chain of the heterodimer Fc, the linker containing the proteolytically cleavable linker, and the second antigen-binding domain Includes, At least one or both of the first and second heavy chains include the third antigen-binding domain or its chain. The multispecific antigen-binding construct according to claim 161.

163. The multispecific antigen-binding construct according to claim 161, wherein the third antigen-binding domain is Fab, and each of the first and second heavy chains comprises the variable heavy (VH) chain and CH1 of Fab.

164. The multispecific antigen-binding construct according to claim 163, further comprising a light chain containing the light chain (VL-CL) of the Fab of the third antigen-binding domain.

165. The third antigen-binding domain is Fab, and the multispecific antigen-binding construct is A first polypeptide comprising the heavy chain variable region (VH) and heavy chain constant region 1 (CH1) of Fab, the heterodimeric immunoglobulin Fc region, and the first antigen-binding domain in order from the N-terminus to the C-terminus, A second polypeptide comprising the VH and CH1 of Fab, the heterodimer Fc region, the linker containing the proteolytically cleavable linker, and the second antigen-binding domain in order from the N-terminus to the C-terminus, A third polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of the aforementioned Fab, A multispecific antigen-binding construct according to any one of claims 157 to 164, comprising:

166. The multispecific antigen-binding construct according to any one of claims 157 to 165, wherein at least one Fc polypeptide of the heterodimer Fc region, optionally each Fc polypeptide comprises at least one amino acid substitution to promote heterodimerization compared to the polypeptide of the homodimer Fc region, and optionally compared to the IgG1 Fc region.

167. The multispecific antigen-binding construct according to claim 166, wherein the one or more amino acid substitutions are knob-into-hole modifications or charge mutations for increasing the electrostatic complementarity of the polypeptide.

168. The first Fc polypeptide in the heterodimer Fc region includes an amino acid substitution selected from Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof, and the second Fc polypeptide in the heterodimer Fc region includes an amino acid substitution T366W, and Optionally, the first Fc polypeptide and the second Fc polypeptide further include amino acid substitutions from non-cysteine ​​residues to cysteine ​​residues. The amino acid substitution of the first polypeptide is at either the Ser354 position or the Y349 position, and the amino acid substitution of the second Fc polypeptide is at the remaining one of the Ser354 position or the Y349 position. The multispecific antigen-binding construct according to claim 166 or claim 167.

169. The multispecific antigen-binding construct according to any one of claims 166 to 168, wherein the first Fc polypeptide comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V, and the second Fc polypeptide comprises the amino acid substitutions S354C and T366W.

170. The multispecific antigen-binding construct according to any one of claims 166 to 169, wherein the first Fc polypeptide comprises the amino acid sequence shown in SEQ ID NO: 100, and the second Fc polypeptide comprises the amino acid sequence shown in SEQ ID NO:

101.

171. The multispecific antigen-binding construct according to any one of claims 166 to 169, wherein the first Fc polypeptide comprises the amino acid sequence shown in SEQ ID NO: 106, and the second Fc polypeptide comprises the amino acid sequence shown in SEQ ID NO:

107.

172. A first polypeptide comprising the heavy chain variable region (VH) and heavy chain constant region (CH1) of Fab, a first immunoglobulin Fc region including the amino acid sequence shown in SEQ ID NO: 106, and a first antigen-binding domain including the amino acid sequence shown in SEQ ID NO: 27, in order from the N-terminus to the C-terminus, A second polypeptide comprising, from the N-terminus to the C-terminus, a second immunoglobulin Fc region containing the VH and CH1 of Fab, the amino acid sequence shown in SEQ ID NO: 107, a linker including the proteolytic cleavage linker shown in SEQ ID NO: 12, and a second antigen-binding domain having at least 95% sequence identity with the sequence shown in any one of SEQ ID NOs: 13-21 and 28-36; A third polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of the aforementioned Fab, A multispecific antigen-binding construct according to any one of claims 157 to 171, comprising:

173. The multispecific antigen-binding construct according to claim 172, wherein the second antigen-binding domain includes the sequence shown in any one of SEQ ID NOs: 13-21 and 28-36.

174. The multispecific antigen-binding construct according to any one of claims 157 to 173, wherein the first target cell antigen is EGFR.

175. The multispecific antigen-binding construct according to claim 174, wherein the third antigen-binding domain is a Fab derived from an antibody selected from the group consisting of necitumumab (11F8), cetuximab, nimotuzumab, and P2X.

176. The aforementioned Fab is, (a) A heavy chain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7 and a light chain containing at least 95% sequence identity with SEQ ID NO: 2 (b) A heavy chain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 205 and a light chain having at least 95% sequence identity with SEQ ID NO: 2 (c) A heavy chain containing an amino acid sequence having at least 95% sequence identity with amino acids 1-217 of SEQ ID NO: 93, and a light chain containing at least 95% sequence identity with SEQ ID NO:

94. (d) A heavy chain containing a sequence having at least 95% sequence identity with amino acids 1-221 of SEQ ID NO: 211 and a light chain containing at least 95% sequence identity with SEQ ID NO: 96, or (e) A heavy chain containing a sequence having at least 95% sequence identity with amino acids 1-217 of SEQ ID NO: 212 and a light chain containing at least 95% sequence identity with SEQ ID NO:

213. A multispecific antigen-binding construct according to claim 174 or claim 175, comprising:

177. The aforementioned Fab is, (a) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2 (b) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 205 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2 (c) A heavy chain containing amino acids 1 to 217 of SEQ ID NO: 93 and a light chain containing the amino acid sequence shown in SEQ ID NO: 94 (d) A heavy chain containing amino acids 1 to 221 of SEQ ID NO: 211 and a light chain containing the amino acid sequence shown in SEQ ID NO: 96, or (e) A heavy chain containing amino acids 1-217 of SEQ ID NO: 212 and a light chain containing the amino acid sequence shown in SEQ ID NO:

213. A multispecific antigen-binding construct according to any one of claims 174 to 176, comprising:

178. The multispecific antigen-binding construct according to any one of claims 174 to 177, wherein the Fab is a necitumumab Fab comprising a heavy chain having at least 95% sequence identity with SEQ ID NO: 7 and a light chain having at least 95% sequence identity with SEQ ID NO:

2.

179. The multispecific antigen-binding construct according to any one of claims 174 to 178, wherein the Fab is a necitumumab Fab comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain containing the amino acid sequence shown in SEQ ID NO:

2.

180. The multispecific antigen-binding construct according to any one of claims 174 to 177, wherein the Fab is a necitumumab Fab comprising a heavy chain having at least 95% sequence identity with SEQ ID NO: 205 and a light chain having at least 95% sequence identity with SEQ ID NO:

2.

181. The multispecific antigen-binding construct according to any one of claims 174 to 177 and 180, wherein the Fab is a necitumumab Fab comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 205 and a light chain containing the amino acid sequence shown in SEQ ID NO:

2.

182. A nucleic acid encoding a multispecific antigen-binding construct according to any one of claims 1 to 181.

183. Each nucleic acid encoding the polypeptide of the construct is a polycistronic sequence, in which each nucleic acid is separated by a multicistronic element. The nucleic acid according to claim 182.

184. The nucleic acid according to claim 183, wherein the multi-cistronic element is a 2A cleavage sequence or an IRES element, and optionally the 2A cleavage sequence is a P2A or T2A sequence.

185. An expression vector comprising the nucleic acid according to any one of claims 182 to 184.

186. A cell comprising the expression vector according to claim 185.

187. A method for producing a multispecific antigen-binding construct, comprising culturing the cells described in claim 186 or a population of such cells under conditions that promote the expression of the multispecific antigen-binding construct from the expression vector by the cells.

188. The method according to claim 187, further comprising isolating the multispecific antigen-binding construct from the cells or population of cells, or from a culture medium in which the cells or population of cells were cultured.

189. A pharmaceutical composition comprising a multispecific antigen-binding construct according to any one of claims 1 to 181 and a pharmaceutically acceptable carrier or excipient.

190. A method for regulating myeloid cell activity on target cells by myeloid cells, comprising contacting a population of target cells with a multispecific antigen-binding construct according to any one of claims 1 to 181 in the presence of myeloid cells in an amount sufficient to regulate myeloid cell activity on the target cells by the myeloid cells.

191. The method according to claim 190, wherein the myeloid cells are macrophages, dendritic cells, monocytes, neutrophils, tumor-associated macrophages (TAMs), tumor-infiltrating macrophages (TIMs), or myeloid-derived immunosuppressive cells (MDSCs).

192. The method according to claim 190 or 191, wherein the target cells are infected with a microorganism or express a microbial antigen.

193. The method according to claim 192, wherein the microbial antigen is a viral antigen.

194. The method according to claim 190 or 191, wherein the cells are cancer cells or the cells express tumor-associated antigens (TAAs).

195. The method according to claim 194, wherein the TAA is selected from the group of TAAs listed in Table 2 or derived from the targets listed in Table 2.

196. A method for treating a subject having a microbial infection, comprising administering to the subject an effective amount of a multispecific antigen-binding construct according to any one of claims 1 to 181 or a pharmaceutical composition according to claim 189, thereby treating the microbial infection in the subject.

197. A method for treating cancer in a subject or delaying its progression, comprising administering to the subject an effective amount of a multispecific antigen-binding construct according to any one of claims 1 to 181 or a pharmaceutical composition according to claim 189, thereby treating the cancer in the subject and / or delaying its progression.

198. The method according to claim 196 or claim 197, wherein the cancer is adenocarcinoma, bile duct (biliary tract) cancer, bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, colon cancer, endometrial cancer, esophageal cancer, glioma, head and neck cancer, head and neck squamous cell carcinoma, leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer, kidney cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, or urothelial carcinoma.

199. The method according to claim 196 or claim 198, further comprising administering an additional therapeutic agent for treating the cancer to the subject.

200. The method according to claim 199, wherein the additional therapeutic agent is a chemotherapeutic agent or a checkpoint inhibitor.

201. A method for treating or delaying the progression of an autoimmune disease or inflammatory disease in a subject, comprising administering to the subject an effective amount of a multispecific antigen-binding construct according to any one of claims 1 to 181 or a pharmaceutical composition according to claim 189, thereby treating and / or delaying the progression of the autoimmune disease or inflammatory disease in the subject.

202. The aforementioned autoimmune diseases or inflammatory diseases include atherosclerosis, obesity, inflammatory bowel disease (IBD), Lyme disease, Hashimoto's thyroiditis, autoimmune uveitis, autoimmune heart valve disease, rheumatoid arthritis, allergic encephalitis, atopic dermatitis, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, Sjögren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylitis, type 1 diabetes mellitus, alopecia areata, vasculitis, and temporal arteritis, graft-versus-host disease (GVHD), asthma, COPD, eosinophilia, conjunctivitis, glomerulonephritis, autoimmune nephritis, paraneoplastic autoimmune diseases, chondritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarthritis-type juvenile rheumatoid arthritis. Polyarticular juvenile rheumatoid arthritis, systemic juvenile juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, myotendonitis, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, fibrous disease, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-joint rheumatoid arthritis, systemic rheumatoid arthritis, enteroarthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa The method according to claim 201, wherein the condition is nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, psoriasis, psoriasis vulgaris, guttate psoriasis, reverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, Crohn's disease, ulcerative colitis, celiac disease, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplant rejection, kidney injury, hepatitis C-induced vasculitis, viral infection, bacterial infection, or spontaneous remission of pregnancy.

203. A method for treating or delaying the progression of a cardiovascular disease in a subject, comprising administering to the subject an effective amount of a multispecific antigen-binding construct according to any one of claims 1 to 181 or a pharmaceutical composition according to claim 189, thereby treating and / or delaying the progression of the cardiovascular disease in the subject.

204. The method according to claim 203, wherein the cardiovascular disease is atherosclerosis, stroke, coronary artery disease, cerebrovascular disease, congenital heart disease, peripheral vascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, cor pulmonale, arrhythmia, endocarditis, myocarditis, eosinophilic myocarditis, valvular heart disease, congenital heart disease, or rheumatic heart disease.

205. A method for treating or delaying the progression of a neurological disorder in a subject, comprising administering to the subject an effective amount of a multispecific antigen-binding construct according to any one of claims 1 to 181 or a pharmaceutical composition according to claim 189, thereby treating and / or delaying the progression of the neurological disorder in the subject.

206. The method according to claim 205, wherein the neurological disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, ophthalmic disorder, glaucoma, myotonic dystrophy, Guillain-Barré syndrome (GBS), myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, traumatic brain injury (TBI), epilepsy, or Huntington's disease (HD).

207. The method according to any one of claims 196 to 206, wherein the subject is a mammal.

208. The method according to claim 207, wherein the mammal is a human, a non-human primate, a farm animal, livestock, or an experimental animal.

209. The method according to any one of claims 196 to 208, wherein the subject is a human.

210. The method according to any one of claims 196 to 209, wherein the multispecific antigen-binding construct is administered orally, rectally, intravenously, intratumorally, or subcutaneously, and more optionally, the multispecific antigen-binding construct is administered subcutaneously or intravenously.

211. A SIRPα-binding molecule comprising at least one heavy-chain-only variable domain (SIRPα VHH domain), which includes a complementarity-determining region 1 (CDR1) containing an amino acid sequence selected from SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, and 45; a complementarity-determining region 2 (CDR2) containing an amino acid sequence selected from SEQ ID NOs: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and a complementarity-determining region 3 (CDR3) containing an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.

212. The at least one SIRPα VHH domain corresponds to, respectively, SEQ ID NOs: 37, 46, and 61, SEQ ID NOs: 38, 46, and 61, SEQ ID NOs: 39, 47, and 62, SEQ ID NOs: 40, 48, and 63, SEQ ID NOs: 41, 49, and 64, SEQ ID NOs: 37, 50, and 61, SEQ ID NOs: 42, 51, and 65, SEQ ID NOs: 43, 52, and 66, SEQ ID NOs: 37, 53, and 67, SEQ ID NOs: 44, 54, and 68, The SIRPα-binding molecule according to claim 211, comprising CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 43, 55, and 63, respectively; SEQ ID NOs: 40, 56, and 69, respectively; SEQ ID NOs: 37, 57, and 70, respectively; SEQ ID NOs: 40, 55, and 63, respectively; SEQ ID NOs: 41, 58, and 71, respectively; SEQ ID NOs: 43, 59, and 72, respectively; SEQ ID NOs: 37, 60, and 73, respectively; or SEQ ID NOs: 45, 56, and 73, respectively.

213. The SIRPα-binding molecule according to claim 211 or claim 212, wherein the SIRPα is human SIRPα.

214. The aforementioned at least one SIRPα VHH domain, The amino acid sequence is one of the sequences shown in any one of SEQ ID NOs: 13-21 and 28-36, or the amino acid sequence is one of the sequences shown in any one of SEQ ID NOs: 13-21 and 28-36 and exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and Binds to SIRPα, A SIRPα-binding molecule according to any one of claims 211 to 213.

215. The SIRPα-binding molecule according to any one of claims 211 to 214, wherein the at least one SIRPα VHH domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 13 to 21 and 28 to 36.

216. The SIRPα-binding molecule according to any one of claims 211 to 215, wherein the binding of the SIRPα VHH domain to SIRPα inhibits or reduces the binding of SIRPα to the differentiated antigen group 47 (CD47).

217. A SIRPα-binding molecule according to any one of claims 211 to 216, wherein the binding affinity of the SIRPα VHH domain to SIRPα is higher than the binding affinity of SIRPα to CD47.

218. The SIRPα-binding molecule according to any one of claims 211 to 217, wherein the VHH domain binds to the IgV domain or a variant of one or more wild-type human SIRPα.

219. The IgV domain or variant of one or more wild-type human SIRPα (i) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103, (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 104, (iii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 105, (iv) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10, and (v) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27 A SIRPα-binding molecule according to any one of claims 211 to 218, selected from among them.

220. The aforementioned VHH is universally reactive, and Wild-type SIRPα, and At least one variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα to improve binding to CD47. A SIRPα-binding molecule according to any one of claims 211 to 219, which binds to the SIRPα molecule.

221. The aforementioned VHH is, (1) IgV domain of wild-type allele SIRPα, optionally, IgV domain of wild-type allele 1 and / or wild-type allele 2 SIRPα, and （２） (a) comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47, and / or (b) A deglycosylated variant, At least one IgV domain of variant SIRPα A SIRPα-binding molecule according to claim 220, which binds to [the specified molecule].

222. The SIRPα-binding molecule according to claim 221, wherein the wild-type human SIRPα optionally comprises, in some cases, the IgV domain of the wild-type human SIRPα, (i) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103, or (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

104.

223. The aforementioned variant SIRPα is optionally configured such that the IgV domain of the aforementioned variant SIRPα is One or more amino acid substitutions in the wild-type SIRPα, selected from the group consisting of L4F or L4I or L4V, V6F or V6I or V6L, V27F or V27I or V27L (A27F or A27I or A27L), I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L or H56R, S66G or S66T or S66A (or L66G or L66T or L66A), K68R, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104). The SIRPα-binding molecule according to claim 221, comprising:

224. The SIRPα-binding molecule according to claim 223, wherein the one or more amino acid substitutions include K53R, E54Q, and S66T (L66T) (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

225. The one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I; or V6I, V27I (or A27I), I31F, E47L, K53R, E54Q, H56P, S66T (or L66T); or L4V, V6I, V27I (or A27I), I31F, E47V, K53R, A SIRPα-binding molecule according to claim 223 or claim 224, wherein the amino acid numbers are E54Q, H56P, V63I, S66T (or L66T), K68R, V92I; or V6I, V27I (or A27I), I31T, E47V, K53R, E54Q, H56P, S66G (or L66G), K68R, V92I, F103V (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

226. The SIRPα-binding molecule according to any one of claims 223 to 225, wherein the one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I (corresponding to the amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104).

227. The SIRPα-binding molecule according to any one of claims 223 to 226, wherein the variant SIRPα is FB3, FD6, FA4, or CV1.

228. The variant SIRPα is optionally configured such that the IgV domain of the variant SIRPα is An amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105, An amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10, or Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27 A SIRPα-binding molecule according to any one of claims 222 to 227, comprising:

229. A binding molecule comprising a SIRPα-binding molecule according to any one of claims 211 to 228 and a second binding domain that binds to a second antigen.

230. The binding molecule according to claim 229, wherein the second antigen is a tumor antigen.

231. The binding molecule according to claim 230, wherein the tumor antigen is a TAA selected from the group of tumor-associated antigens (TAAs) listed in Table 2, or a TAA derived from a target listed in Table 2.

232. The binding molecule according to claim 230, wherein the tumor antigen is CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), EGFR, HER2, CD117, C-Met, PTHR2, HAVCR2 (TIM3).

233. The binding molecule according to any one of claims 229 to 231, wherein the binding domain that binds to the second antigen is an antibody or an antigen-binding fragment.

234. The binding molecule according to any one of claims 229 to 233, wherein the binding molecule is a bispecific antibody.

235. A nucleic acid encoding a SIRPα-binding molecule according to any one of claims 211 to 228.

236. A nucleic acid encoding the binding molecule described in any one of claims 229 to 234.

237. An expression vector comprising the nucleic acid according to claim 235 or claim 236.

238. A cell comprising the expression vector described in claim 237.

239. A method for producing a SIRPα-binding molecule, comprising culturing the cells described in claim 231 or a population of such cells under conditions that promote the expression of the SIRPα-binding molecule from the expression vector by the cells.

240. The method according to claim 239, further comprising isolating the SIRPα-binding molecule from the cells or a group of cells, or from a culture medium in which the cells or a group of cells were cultured.

241. A pharmaceutical composition comprising a SIRPα-binding molecule according to any one of claims 211 to 228 or a binding molecule according to any one of claims 229 to 234, and a pharmaceutically acceptable carrier or excipient.

242. A method for treating cancer in a subject or delaying its progression, comprising administering to the subject an effective amount of a SIRPα-binding molecule according to any one of claims 211 to 228, a binding molecule according to any one of claims 229 to 234, or a pharmaceutical composition according to claim 241, thereby treating the cancer in the subject and / or delaying its progression.

243. The method according to claim 242, wherein the cancer is adenocarcinoma, bile duct (biliary tract) cancer, bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, colon cancer, endometrial cancer, esophageal cancer, glioma, head and neck cancer, head and neck squamous cell carcinoma, leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer, kidney cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, urogenital cancer, or urothelial carcinoma.

244. The method according to claim 242 or claim 243, further comprising administering an additional therapeutic agent to the subject for treating the cancer.

245. The method according to claim 244, wherein the additional therapeutic agent is a chemotherapeutic agent or a checkpoint inhibitor.

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