Therapeutic binding agents for conditionally promoting myeloid cell activity against target cells and uses thereof

Abstract

The present disclosure provides, inter alia, therapeutic binding agents that can direct the activity of myeloid cells to a target population of cells, such as cancer cells.

Description

Cross-references to related applications

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

[0002] The sequence list is incorporated by reference. This application is submitted together with an electronic sequence list. The sequence list is provided as a document named 336402000140SeqList.XML, created on August 12, 2024, and is 461,658 bytes in size. The information in the electronic sequence list is incorporated herein by reference in its entirety. Background Technology

[0003] Myeloid cells are immune cells found in almost all tissues, contributing to immune responses and cell maintenance. In the tumor setting, myeloid cells are the most abundant type of immune cell and perform diverse activities. Among their potential functions, myeloid cells, such as macrophages, granulocytes, monocytes, and dendritic cells (DCs), can recognize cancer cells and / or contribute to anti-tumor responses (e.g., by phagocytosing cancer cells). Summary of the Invention

[0004] This disclosure particularly provides therapeutic binders that can direct the activity of myeloid cells to a target cell population, such as by directly killing unwanted cells (such as cancer cells) or target immune cells, indirectly killing unwanted cells (such as cancer cells) or target immune cells, or inhibiting target cells (such as target immune cells). This disclosure particularly provides therapeutic binders that can lead to the direct or indirect elimination of target cells, for example, by directing the activity of myeloid cells (e.g., phagocytic activity of myeloid cells) to a target cell population, by antibody-dependent cytotoxicity (ADCC), and / or by the activity of the therapeutic binder that directly kills target cells. In various embodiments, the target cells may be, for example, cancer cells or target immune cells. In some embodiments, this disclosure also provides such therapeutic binders that selectively direct myeloid cells to target a cell population (such as cancer cells) in specific situations (such as in a tumor setting). In some embodiments, the therapeutic binders covered by this disclosure direct the elimination of target cells through a combination of elements, for example, by directing the activity of myeloid cells (e.g., phagocytic activity) to a target cell population in a specific environment (e.g., to cancer cells in a tumor environment, autoimmune cells in an inflammatory environment, etc.). This combination of elements may include: (1) at least one binding domain that binds an antiphagocytic protein (APP) (e.g., expressed on myeloid cells or on cells targeted for killing or inhibiting adverse activity) (APP binding domain), (2) at least one binding domain that conditionally inhibits the interaction of the anti-APP binding domain with its target (masking domain), and (3) a proteolytically cleavable linker whose localization allows its cleavage to release the inhibition of APP binding by the masking domain. The therapeutic binders covered by this disclosure may include one or more additional elements, such as one or more elements that promote and / or activate myeloid cells, and / or at least one binding domain that binds a target cell antigen (target cell binding domain). In some embodiments, the proteolytically cleavable linker is cleaved by proteases characteristic and / or specific to a particular target environment (e.g., the tumor microenvironment), thereby achieving activatable activity. This disclosure includes the finding that therapeutic binders according to this disclosure can selectively guide the elimination of target cells, for example, by directing myeloid cells in the tumor environment to the target and / or phagocytosing target cells, through antibody-dependent cytotoxicity (ADCC), and / or through the activity of the therapeutic binder by directly killing target cells. In various embodiments, the target cells are cancer cells, and the therapeutic binders provided herein can be used to treat cancer. In various embodiments, the therapeutic binders provided herein modulate the activity of target cells in a particular target environment (e.g., inhibiting adverse activities, such as inhibiting inflammatory and / or autoimmune functions of immune cell subtypes and / or antigen-specific immune cells).

[0005] The methods and compositions covered by this disclosure are associated with numerous advantages. For example, this disclosure understands that APP blockade can direct the activity (e.g., phagocytic activity) of myeloid cells to target cells (such as cancer cells), but such blockade may result in myeloid cells in healthy tissue targeting and destroying healthy cells, with harmful and / or toxic effects. This challenge is particularly acute because many APPs are present on both normal cells and target cells (such as cancer cells) (e.g., ubiquitous). The various therapeutic binders covered by this disclosure comprise a masking domain that associates with a proteolytically cleavable linker, the masking domain being positioned such that it inhibits the binding of the APP-binding domain to the APP until this inhibition is lifted by cleavage of the linker. Thus, the various therapeutic binders covered by this disclosure advantageously provide selective targeting of APP-expressing (e.g., myeloid) cells in a target environment (e.g., tumor microenvironment) to direct myeloid cell activity (e.g., phagocytic activity) against target cells (such as cancer cells). This selective targeting of myeloid cells in the target environment does not damage healthy cells and improves the pharmacokinetic properties (e.g., therapeutic index), safety, and / or efficacy against the target cells (e.g., cancer cells).

[0006] In some embodiments, the therapeutic binders of this disclosure cause the elimination of target cells via antibody-dependent phagocytosis (ADCP). In some embodiments, the therapeutic binders of this disclosure cause the elimination of target cells via antibody-dependent cytotoxicity (ADCC). In some embodiments, the therapeutic binders of this disclosure cause the elimination of target cells through direct killing (also referred to herein as direct elimination). In some embodiments, the therapeutic binders covered by this disclosure guide or promote the activity of myeloid cells (e.g., conditionally guide or promote the activity of myeloid cells against a target cell population), the activity being selected from the group consisting of: phagocytosis (e.g., antibody-dependent phagocytosis (ADCP)), cell-mediated direct killing activity (e.g., antibody-dependent cytotoxicity (ADCC), lymphocyte-promoting cytotoxic activity, etc.), and indirect cytotoxic activity (e.g., promoting complement-dependent cytotoxicity (CDC), reducing lymphocyte immune checkpoint suppression, reducing myeloid cell immune checkpoint suppression, etc.). In some embodiments, the therapeutic binders covered by this disclosure direct or promote specific activities of myeloid cells (e.g., conditionally directing or promoting myeloid cell activity against a target cell population), such as 1) phagocytosis, rather than cell-mediated direct or indirect cell-killing activity; 2) cell-mediated direct cell-killing activity, rather than phagocytosis or indirect cell-killing activity; and 3) indirect cell-killing activity, rather than phagocytosis or cell-mediated direct cell-killing activity. In some embodiments, the therapeutic binders covered by this disclosure modulate myeloid cell activity against target cells (e.g., conditionally directed activity of myeloid cells against a target cell population), which involves inhibiting undesirable activities, such as inhibiting the pro-inflammatory and / or autoimmune functions of immune cell subtypes and / or antigen-specific immune cells. In some embodiments, the therapeutic binders covered by this disclosure modulate myeloid cell activity against target cells (e.g., conditionally directed activity of myeloid cells against a target cell population), which involves promoting desired activities, 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 some aspects, this 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 antigen-binding domain and the second antigen-binding domain are connected by a protein-cleavable linker.

[0008] In at least some aspects, this 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 antigen-binding domain and the second antigen-binding domain are connected by a proteolytically cleavable linker, wherein 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 APP, and wherein when the linker has been 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 some aspects, this disclosure provides a multispecific antigen-binding construct comprising: (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 comprising a proteolytically cleavable linker; (iv) a third antigen-binding domain that binds to a first target cell antigen; and (v) an immunoglobulin Fc region comprising a linker that connects the second antigen-binding domain to the first antigen-binding domain or the immunoglobulin Fc region.

[0010] In various embodiments, the first antigen-binding domain and the second antigen-binding domain are connected via a connector comprising a protein-cleavable linker. In various embodiments, the immunoglobulin Fc region and the second antigen-binding domain are connected via a connector comprising a protein-cleavable linker. In various embodiments, 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 APP, and when the linker has been protein-cleaved, the second antigen-binding domain does not interfere with the binding of the first antigen-binding domain to APP.

[0011] In various embodiments, the APP is selected from the group consisting of or including: differentiation cluster 47 (CD47), differentiation cluster 24 (CD24), programmed cell death 1 ligand 1 (PD-L1), programmed cell death 1 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). In various embodiments, the first antigen-binding domain 1) inhibits the interaction of the APP with its binding partner on myeloid cells, or 2) inhibits the interaction of the APP with its binding partner on cells targeted to regulate (e.g., kill, e.g., through phagocytosis). In various implementation schemes, myeloid cells are macrophages, dendritic cells, monocytes, neutrophils, tumor-associated macrophages (TAMs), tumor-infiltrating macrophages (TIMs), or myeloid-derived suppressor cells (MDSCs). In various embodiments, the first antigen-binding domain inhibits interactions selected from or comprising the group consisting of: the interaction between CD47 and SIRPα, the interaction between CD24 and SIGLEC10, the interaction between PD-1 and PD-1 ligands (PD-L1 or PD-L2), the interaction between LILRB1 ligands (β2M or MHC-I complex) and LILRB1, and the interaction between LILRB2 ligands and LILRB2, optionally wherein the first antigen-binding domain is SIRPα or a domain or fragment thereof, SIGLEC10 or a domain or fragment thereof, PD-1 or a domain or fragment thereof, LILRB1 or a domain or fragment thereof, LILRB2 or a domain or fragment thereof, PD-L1 or a domain or fragment thereof, PD-L2 or a domain or fragment thereof, CD47 or a domain or protein thereof, CD24 or a domain or protein thereof, β2M or a domain or protein thereof, or a protein of the MHC-I complex (HLA-A, HLA-B, or HLA-C).

[0012] In various embodiments, APP is CD47. In various embodiments, a first antigen-binding domain binds to CD47 and inhibits the interaction of CD47 with wild-type SIRPα, optionally wherein the first antigen-binding domain binds to wild-type cell surface-expressed CD47 and inhibits the interaction of wild-type cell surface-expressed CD47 with wild-type cell surface-expressed SIRPα. In various embodiments, the first antigen-binding domain comprises (i) an 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, which is engineered to improve binding to APP. In various embodiments, the cell surface-expressed protein is wild-type SIRPα, and the first antigen-binding domain comprises (i) an 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, which is engineered to improve binding to APP, wherein APP is CD47. In various embodiments, the binding fragment includes the immunoglobulin variable (V) region (domain 1) of the ECD (optionally the ECD of wild-type SIRPα) of a protein expressed on the cell surface. In various embodiments, the first antigen-binding domain includes a domain of wild-type SIRPα that binds CD47, or a variant thereof, which includes one or more amino acid substitutions in the wild-type SIRPα domain that improve binding to CD47.

[0013] In at least some aspects, this disclosure provides a multispecific antigen-binding construct comprising: a) a first antigen-binding domain comprising (i) a domain of wild-type SIRPα binding to an antiphagocytic protein (APP), or (ii) a variant thereof comprising one or more amino acid substitutions in the wild-type SIRPα domain that improve binding to APP, wherein APP is CD47; and b) a second antigen-binding domain, which is an anti-SIRPα antibody or an 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 antigen-binding domain and the second antigen-binding domain are connected by a linker comprising a proteolytically cleavable linker. In various embodiments, the first antigen-binding domain is 100 to 120 amino acids in length, optionally 106 to 118 amino acids, and more preferably 112 to 118 amino acids. 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 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. 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 or comprises the sequence shown in SEQ ID NO: 103.

[0014] 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 or comprises the sequence shown in SEQ ID NO: 104.

[0015] In various embodiments, the first antigen-binding domain is a variant SIRPα that includes one or more amino acid substitutions in the IgV domain of wild-type SIRPα to improve binding to CD47. In various embodiments, the variant SIRPα binds to wild-type human CD47, whose dissociation constant (K0) is... D The values ​​are below 100 nanomoles (nM), below 10 nM, below 1 nM, below 100 picomoles (pM), below 10 pM, or below 1 pM, or any combination of the foregoing values. In various embodiments, the variant SIRPα binds to wild-type human CD47, whose dissociation constant (Ki) is... DThe concentration is below 100 nanomolars (nM), optionally 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. In each embodiment, the variant SIRPα binds to wild-type human CD47, whose dissociation constant (Ki) is... D Below 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. In various embodiments, the variant SIRPα binds to wild-type human CD47, whose dissociation constant (Ki) is... D The value is less than 100 picomoles (pM). In various embodiments, the variant SIRPα binds to wild-type human CD47, whose dissociation constant (K0.05) is less than 100 picomoles (pM). D The range is 1pM to 100pM, optionally 1pM to 75pM, 1pM to 50pM, 1pM to 25pM, 1pM to 10pM, 10pM to 100pM, 10pM to 75pM, 10pM to 50pM, 10pM to 25pM, 25pM to 100pM, 25pM to 75pM, 25pM to 50pM, or 50pM to 100pM, 50pM to 75pM, or 75pM to 100pM.

[0016] 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 numbers of SEQ ID NO: 103 or SEQ ID NO: 104. In each embodiment, at least one amino acid substitution is E54Q, corresponding to the amino acid number of SEQ ID NO: 103 or SEQ ID NO: 104.

[0017] In various embodiments, one or more amino acid substitutions include K53R, E54Q, and S66T (L66T), corresponding to the amino acid numbers 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 A27I), 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 amino acid numbers in 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 amino acid numbers in 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 or comprises the amino acid sequence shown in SEQ ID NO: 105.

[0018] 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 number 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: 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 comprises the amino acid sequence shown in SEQ ID NO: 10.

[0019] 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 or comprises the amino acid sequence shown in SEQ ID NO: 27.

[0020] 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, Fab, F(ab')2, Fab', dsFv, Fde, or sdFv. In various embodiments, the first antigen-binding domain is a single-domain antibody, which is a VHH. In various embodiments, the VHH is a camel heavy chain antibody, a humanized VHH domain, an affinity-matured VHH domain, or a human VHH domain. 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: 208. In various embodiments, the first antigen binding comprises the sequence shown in SEQ ID NO: 208. In various embodiments, the dissociation constant of the second antigen-binding domain to the first antigen-binding domain is higher than the dissociation constant of the first antigen-binding domain to APP. In various embodiments, the dissociation constant (Kd) of the second antigen-binding domain 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 that of the first antigen-binding domain to APP. In various embodiments, when the cleavable adapter is cleaved, the second antigen-binding domain does not interfere with or compete for the binding of the first antigen-binding domain to APP. In various embodiments, the dissociation constant of the second antigen-binding domain to the first antigen-binding domain is 1 nM or higher, optionally 100 nM to 1 µM, 10 nM to 1 µM, or 1 nM to 1 µM. In various embodiments, the dissociation constant of the second antigen-binding domain to the first antigen-binding domain is 1 nM or higher. In various embodiments, the dissociation constant of the second antigen-binding domain to the first antigen-binding domain is 10 nM or higher. In various embodiments, the dissociation constant of the second antigen-binding domain to the first antigen-binding domain is 100 nM or higher. In various embodiments, the dissociation constant of the second antigen-binding domain to the first antigen-binding domain is 1 µM or higher.

[0021] In various embodiments, the second antigen-binding domain is an antibody or an antigen-binding fragment. In various embodiments, the second antigen-binding domain is an anti-SIRPα antibody or an 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, which is a VHH. In various embodiments, the VHH is a camel heavy chain antibody, a humanized VHH domain, an affinity-matured VHH domain, or a human VHH domain.

[0022] In various embodiments, VHH comprises: complementarity-determining region 1 (CDR1) containing amino acid sequences selected from SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44 and 45; complementarity-determining region 2 (CDR2) containing amino acid sequences selected from SEQ ID NO: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60; and complementarity-determining region 3 (CDR3) containing amino acid sequences selected from SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73. In various implementations, the VHH domain includes CDR1, CDR2, and CDR3 as follows: SEQ ID NO: 37, 46, and 61; SEQ ID NO: 38, 46, and 61; SEQ ID NO: 39, 47, and 62; SEQ ID NO: 40, 48, and 63; SEQ ID NO: 41, 49, and 64; SEQ ID NO: 37, 50, and 61; SEQ ID NO: 42, 51, and 65; SEQ ID NO: 43, 52, and 66; SEQ ID NO: 37, 53, and 67; SEQ ID NO: 44, 54, and 68; SEQ ID NO: 43, 55, and 63; SEQ ID NO: 40, 56, and 69; SEQ ID NO: 37, 57, and 70; SEQ ID NO: 40, 55, and 63; SEQ ID NO: 41, 58, and 71; 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. In various embodiments, the VHH domain comprises the amino acid sequence shown in any 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 of SEQ ID NOs: 13-21 and 28-36, and binds SIRPα. In various embodiments, the VHH domain comprises the amino acid sequence shown in any of SEQ ID NOs: 13-21 and 28-36.

[0023] 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 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 to improve 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 to improve binding to CD47. In various embodiments, the IgV domain of one or more wild-type human SIRPα or variants thereof is selected from: (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 SEQ ID NO: 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 SEQ ID NO: 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: 105; 10. 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; and (v) 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.

[0024] In various embodiments, the anti-SIRPα antibody or antigen-binding fragment binds to (1) the IgV domain of the wild-type allele SIRPα (optionally wild-type allele 1 and / or wild-type allele 2 SIRPα), and (2) at least one IgV domain of a variant SIRPα that includes one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improve binding to CD47. In various embodiments, the anti-SIRPα antibody or antigen-binding fragment binds to the IgV domain of wild-type human SIRPα, optionally wild-type human SIRPα, wherein the wild-type human SIRPα comprises: (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, optionally wild-type human SIRPα comprising 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, optionally wild-type human SIRPα comprising the amino acid sequence shown in SEQ ID NO: 104.

[0025] In various embodiments, the anti-SIRPα antibody or antigen-binding fragment binds to the IgV domain of the variant SIRPα, optionally the variant SIRPα, wherein the variant SIRPα contains one or more amino acid substitutions selected from the group consisting of wild-type 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, L66T, or L66A), K68R, V92F, V92I, or V92L, F94I, F94L, or F94V, and F103I, F103L, or F103V, corresponding to amino acid numbers in 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 amino acid numbers in SEQ ID NO: 103 or SEQ ID NO: 104.

[0026] 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 A27I), 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 amino acid numbers in 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 amino acid numbers in SEQ ID NO: 103 or SEQ ID NO: 104. In various embodiments, variant SIRPα is named FB3, FD6, FA4, or CV1.

[0027] In various embodiments, the anti-SIRPα antibody or antigen-binding fragment binds to the IgV domain of variant SIRPα, optionally variant SIRPα, which comprises: (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; (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: 10; 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. 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.

[0028] In each implementation scheme, the dissociation constant (Ka) of the anti-SIRPα antibody or antigen-binding fragment binding to wild-type human SIRPα or its variants is specified. DThe KD of wild-type human SIRPα or a variant thereof with wild-type human CD47 is at least 2, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times that of wild-type human CD47, optionally wherein CD47 is CD47 expressed on the cell surface.

[0029] In various embodiments, the linker is a substrate of the protease, optionally wherein the protease is an extracellular protease, and / or the linker is a substrate of renin, pepsin C, aspartic protease A, matrix metalloproteinase (MMP), matrix protease, urokinase-type plasminogen activator (uPA), integrin metalloproteinase (ADAM), integrin metalloproteinase containing a platelet-reactive protein motif (ADAMTS), podocyte oleoresin, urokinase, or hepatic serine.

[0030] In various embodiments, the proteolytically cleavable linker is a polypeptide that functions as a substrate of a 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 matrix proteases, matrix metalloproteinases (MMPs), granzyme B, and combinations thereof. In various embodiments, the protease is a matrix protease. In various embodiments, the proteolytically cleavable linker is VHMPLGFLGPRQARVVN (SEQ ID NO: 22). In various embodiments, the linker comprising the proteolytically cleavable linker comprises an N-terminal and / or C-terminal GS linker sequence. In various embodiments, the GS linker sequence is sequence (GGGGS)n, where n is 1 to 5 (SEQ ID NO: 259), optionally wherein the GS linker sequence is GGGGSGGGGS (SEQ ID NO: 9) or GGGGS (SEQ ID NO: 11).

[0031] In various embodiments, the construct further includes a third antigen-binding domain that binds to a first target cell antigen, optionally wherein the first target cell antigen is expressed on cells targeted to regulate (e.g., kill, such as through phagocytosis). In various embodiments, the first target cell antigen is expressed on cells targeted to be acted upon by myeloid cell activity. In various embodiments, the third antigen-binding domain is an antibody or an antigen-binding fragment. In various embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv), sc(Fv)2, Fab, Fv, Fav, F(ab')2, Fab', dsFv, Fde, sdFv, or a single-domain antibody (sdAb).

[0032] In various embodiments, the first target cell antigen is a microbial antigen, a peptide-major histocompatibility complex (pMHC), or a tumor-associated antigen (TAA), optionally wherein the TAA is selected from the group of TAAs listed in Table 2 or derived from the targets listed in Table 2. In various embodiments, 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.

[0033] 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-matured 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 comprises two distinct single-chain antibody fragments. In various embodiments, the third antigen-binding domain is doubly complementary. In various embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain are antibodies. In various implementation schemes, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain include: the extracellular domain (ECD) of a cell surface-expressed protein, a fragment of the ECD of a cell surface-expressed protein, a mutant form (variant) of the ECD of a cell surface-expressed protein engineered to improve binding to the target, the binding domain of an antibody, a functional fragment of an antibody, its variable domain, VH domain, VL domain, VNAR domain, VHH domain, single-chain variable fragment (scFv), sc(Fv)2, Fab, Fv, Fav, F(ab')2, Fab', dsFv, Fde, sdFv, single-domain antibody (sdAb), nano... Rice antibodies, bispecific antibodies, biantibodies, intracellular antibodies, domain antibodies, antibody mimics, zybody, peptide-Fc fusions, camel antibodies, camel-derived antibodies, masking antibodies, affybody, anti-idiotypic (anti-Id) antibodies, single-chain biantibodies, tandem biantibodies, VHH, anti-carrier proteins, small antibodies, BiTE, ankyrin repeat proteins, DARPIN, avimer, DART, TCR-like antibodies, adnectin, affilin, transmembrane antibodies, affibody, TrimerX, microproteins, fynomer, centyrin, KALBITOR, CAR, engineered TCR, or functional fragments or combinations thereof. In various embodiments, the antibody includes a constant domain of immunoglobulins selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. In various embodiments, the construct and / or antibody includes a constant domain derived from human immunoglobulins.

[0034] In various embodiments, the multispecific antigen-binding construct further includes a fourth antigen-binding domain that binds to a second target cell antigen, optionally wherein the second target cell antigen is expressed on cells targeted for regulation (e.g., killing, such as through phagocytosis). In various embodiments, the fourth antigen-binding domain is an antibody or an antigen-binding fragment. In various embodiments, the antibody or antigen-binding fragment is a single-chain 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, and the TAA is selected from the group of TAAs listed in Table 2 or derived from the targets listed in Table 2.

[0035] 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.

[0036] In various embodiments, the Fc region is a variant Fc region containing one or more amino acid mutations or substitutions that enhance the antibody-dependent cytotoxicity (ADCC)-promoting activity and / or antibody-dependent phagocytosis (ADCP)-promoting activity of the multispecific antigen-binding construct. In various embodiments, with wild-type human IgG1...Compared to the Fc region, the variant Fc region contains one or more amino acid mutations. In various embodiments, the variant Fc region contains mutations at one or more positions selected from the group consisting of: 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, 2 97, 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, wherein optionally one or more of the mutations are substitutions, and further optionally wherein the substitutions are selected from the group consisting of or composed of: 220S, 229S, 232G, 233P, 234A, 234D, 234E, 234F, 234G, 234 H, 234L, 234N, 234Q, 234T, 234V, 234Y, 235A, 235D, 235E, 235F, 235G, 235H, 235N, 235P, 235Q, 235R, 235S, 235T, 235W, 235Y, 236A, 236E, 23 6I, 236N, 236P, 236R, 237A, 237K, 237L, 237N, 237P, 238K, 238S, 239D, 239E, 239F, 239H, 239N, 239Q, 239R, 239T, 239Y, 240M, 240T, 241A, 2 41E, 241L, 241W, 241Y, 243L, 243Q, 243R, 243W, 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, 305I, 309L, 316D, 318A, 324T, 325A, 325E, 325H, 325L, 325Q, 325T, 325V, 326W, 327G, 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, 331D, 331E, 331F, 331G, 331H, 331K, 331L, 331M, 331N, 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 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 contains 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 a 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 a 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 N-terminus to C-terminus, a heavy chain variable region (VH) and a 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 a light chain constant region (CL) of Fab. In various embodiments, the multispecific polypeptide construct comprises two identical first polypeptides and two identical second polypeptides, wherein the two first polypeptides are covalently linked by disulfide bonds, and wherein each of the second polypeptides is covalently linked to one of the first polypeptides by a disulfide bond. In various embodiments, In various embodiments, the third antigen-binding region is Fab, and the multispecific antigen-binding construct comprises: a first polypeptide comprising, from N-terminus to 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 the proteolytically cleavable linker shown in SEQ ID NO: 12, and a second antigen-binding domain comprising a sequence having at least 95% sequence identity with the sequences shown in any of SEQ ID NO: 13-21 and 28-36; and a second polypeptide comprising a light chain variable region (VL) and a 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 N-terminus to 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 the proteolytically cleavable linker shown in SEQ ID NO: 12, and a second antigen-binding domain comprising a sequence having at least 95% sequence identity with the sequences shown in any one of SEQ ID NO: 13-21 and 28-36; and a second polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of Fab.

[0038] In various embodiments, the second antigen-binding domain comprises the sequence shown in any one of SEQ ID NO: 13-21 and 28-36. In various embodiments, the multispecific antigen-binding construct comprises a peptide linker located 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 to 5 (SEQ ID NO: 259). In various embodiments, the GS linker is GGGGS (SEQ ID NO: 11), GGGGSGGGGS (SEQ ID NO: 9), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 23), or GGGGSGGGGSGGGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 24).

[0039] In each embodiment, the first target cell antigen is EGFR. In each embodiment, the third antigen-binding domain is a Fab derived from an antibody selected from the group consisting of nexituzumab (11F8), cetuximab, nimotuzumab, and P2X. In various embodiments, Fab comprises: (a) a heavy chain comprising 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 comprising 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 comprising 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 comprising 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 comprising 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: 205. 213 is a light chain having at least 95% sequence identity. In various embodiments, Fab comprises: (a) 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; (b) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 205 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 2; (c) a heavy chain comprising amino acids 1-217 of SEQ ID NO: 93 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 94; (d) a heavy chain comprising amino acids 1-221 of SEQ ID NO: 211 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 96; or (e) a heavy chain comprising amino acids 1-217 of SEQ ID NO: 212 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 213.

[0040] In various embodiments, Fab is a nexituzumab Fab comprising: a heavy chain comprising a 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. In various embodiments, Fab is a nexituzumab Fab comprising: 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 various embodiments, wherein Fab is a nexituzumab Fab, comprising: a heavy chain comprising a 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. In various embodiments, Fab is a nexituzumab Fab comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 205, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 2.

[0041] In various embodiments, the multispecific polypeptide construct comprises: a first polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in any of SEQ ID NO: 110-120, and a second polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 2. In various embodiments, the multispecific polypeptide construct comprises: a first polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in any of SEQ ID NO: 110-120, and a second polypeptide chain comprising the sequence shown in SEQ ID NO: 2. In various embodiments, the multispecific polypeptide construct comprises: a first polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in any of SEQ ID NO: 125-137, and a second polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 2. In various embodiments, the multispecific polypeptide construct comprises: a first polypeptide chain comprising a sequence shown in any of SEQ ID NO: 125-137, and a second polypeptide chain comprising the sequence shown in SEQ ID NO: 2.

[0042] In various embodiments, the third antigen-binding region is a single-chain antibody fragment, and the multispecific antigen-binding construct comprises a polypeptide including a third antigen-binding region, an Fc region, a first antigen-binding domain, a linker including a protein-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, from the N-terminus to the C-terminus, sequentially includes a third antigen-binding region, an Fc region, a first antigen-binding domain, a linker including a protein-cleavable linker, and a second antigen-binding domain.

[0043] In various embodiments, the immunoglobulin Fc region is a variant Fc region comprising a modified hinge domain containing a substitution of the amino acid EPKSC for 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.

[0044] In various embodiments, the first target cell antigen is EGFR. In various embodiments, the third antigen-binding domain is an scFv derived from the group consisting of nexituzumab (11F8), cetuximab, nimotuzumab, and P2X. In various embodiments, the third antigen-binding domain is an scFv derived from nexituzumab (11F8). In various embodiments, the scFv comprises: a VH chain containing an amino acid sequence having at least 95% sequence identity with the variable heavy (VH) chain sequence presented in SEQ ID NO: 207, and a VL chain containing an amino acid sequence having at least 95% sequence identity with the variable light (VL) chain sequence presented in SEQ ID NO: 207. In various embodiments, the scFv contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 207. In various embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 207.

[0045] In various embodiments, the Fc region is a heterodimeric Fc region. In various embodiments, either the first antigen-binding domain or the third antigen-binding domain is divalent, and the other of the first and third antigen-binding domains is 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.

[0046] In various embodiments, the multispecific antigen-binding construct comprises: a first antigen-binding domain binding to an antiphagocytic protein (APP), a second antigen-binding domain binding 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 serving as a target cell antigen-binding domain, binding to a target cell antigen expressed on a cell targeted for myeloid cell activity, wherein the second antigen-binding domain is linked to one of the first Fc polypeptide or the second Fc polypeptide via a proteolytically cleavable linker. In various embodiments, the multispecific antigen-binding construct comprises (1) a first heavy chain comprising a first polypeptide chain containing a heterodimeric Fc and a first antigen-binding domain; and (2) a second heavy chain comprising a second polypeptide chain containing a heterodimeric Fc, a linker containing a proteolytically cleavable linker, and a second antigen-binding domain, wherein at least one or both of the first and second heavy chains comprise a third antigen-binding domain or a chain thereof. 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 of Fab and CH1. In various embodiments, the multispecific antigen-binding construct further comprises a light chain comprising a light chain (VL-CL) of Fab of the third antigen-binding domain.

[0047] In various embodiments, when the third antigen-binding domain is Fab, the multispecific antigen-binding construct comprises: a first polypeptide comprising, from N-terminus to C-terminus, a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1) of Fab, a first polypeptide of the heterodimeric immunoglobulin Fc region, and a first antigen-binding domain; a second polypeptide comprising, from N-terminus to C-terminus, VH and CH1 of Fab, a second polypeptide of the heterodimeric Fc region, a linker comprising a proteolytically cleavable linker, and a second antigen-binding domain; and a third polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of Fab.

[0048] In various embodiments, compared to the homodimeric Fc region and optionally compared to the IgG1 Fc region, at least one Fc polypeptide in the heterodimeric Fc region, optionally each Fc polypeptide, comprises at least one amino acid substitution to promote heterodimerization. In various embodiments, one or more amino acid substitutions are mortar-and-mortar modifications or charge mutations to enhance the electrostatic complementarity of the polypeptide. In various embodiments, the first Fc polypeptide in the heterodimeric Fc region comprises an amino acid substitution selected from Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof, and the second Fc polypeptide in the heterodimeric Fc region comprises the amino acid substitution T366W, and optionally, the first and second Fc polypeptides further comprise amino acid substitutions that change non-cysteine ​​residues to cysteine ​​residues, wherein the amino acid substitution of the first polypeptide is located at one of Ser354 and Y349, and the amino acid substitution of the second Fc polypeptide is located at the other of Ser354 and Y349. In various embodiments, the first Fc polypeptide comprises amino acid substitutions Y349C, T366S, L368A, and Y407V, and the second Fc polypeptide comprises 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.

[0049] In various embodiments, the multispecific antigen-binding construct comprises: a first polypeptide comprising, from N-terminus to C-terminus, a heavy chain variable region (VH) and a heavy chain constant region (CH1) of Fab, a first immunoglobulin Fc region comprising the amino acid sequence shown in SEQ ID NO: 106, and a first antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 27; a second polypeptide comprising, from N-terminus to C-terminus, VH and CH1 of Fab, a second immunoglobulin Fc region comprising the amino acid sequence shown in SEQ ID NO: 107, a linker comprising the proteolytically cleavable linker shown in SEQ ID NO: 12, and a second antigen-binding domain comprising a sequence having at least 95% sequence identity with the sequences shown in any one of SEQ ID NO: 13-21 and 28-36; and a third polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of Fab. In various embodiments, the second antigen-binding domain comprises the sequence shown in any one of SEQ ID NO: 13-21 and 28-36.

[0050] In each embodiment, the first target cell antigen is EGFR. In each embodiment, the third antigen-binding domain is a Fab derived from an antibody selected from the group consisting of nexituzumab (11F8), cetuximab, nimotuzumab, and P2X. In various embodiments, Fab comprises: (a) a heavy chain comprising 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 comprising 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 comprising 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 comprising 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 comprising 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: 205. 213 is a light chain having at least 95% sequence identity. In various embodiments, Fab comprises: (a) 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; (b) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 205 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 2; (c) a heavy chain comprising amino acids 1-217 of SEQ ID NO: 93 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 94; (d) a heavy chain comprising amino acids 1-221 of SEQ ID NO: 211 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 96; or (e) a heavy chain comprising amino acids 1-217 of SEQ ID NO: 212 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 213.

[0051] In various embodiments, Fab is a nexituzumab Fab comprising: a heavy chain comprising a 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. In various embodiments, Fab is a nexituzumab Fab comprising: 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 various embodiments, Fab is a nexituzumab Fab comprising: a heavy chain comprising a 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. In various embodiments, wherein Fab is a nexituzumab Fab comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 205, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 2.

[0052] In at least some aspects, this disclosure provides nucleic acids encoding multispecific antigen-binding constructs according to this disclosure. In at least some aspects, this disclosure provides expression vectors comprising the nucleic acids of this disclosure. In at least some aspects, this disclosure provides cells comprising the expression vectors of this disclosure. In at least some aspects, this disclosure provides a method for producing multispecific antigen-binding constructs, the method comprising culturing cells of this disclosure or populations of such cells under conditions conducive to the expression of the multispecific antigen-binding constructs from the expression vectors. In various embodiments, the method for producing multispecific antigen-binding constructs as provided herein further includes isolating the multispecific antigen-binding constructs from cells or cell populations, or from a culture medium in which the cells or cell populations are cultured. In at least some aspects, this disclosure provides a pharmaceutical composition comprising the multispecific antigen-binding construct of this disclosure and a pharmaceutically acceptable carrier or excipient.

[0053] In at least some aspects, this disclosure provides a method for enhancing the regulation of target cells by myeloid cells (e.g., killing, such as through phagocytosis), the method comprising: contacting a population of target cells with an amount sufficient to regulate or enhance the regulation of target cells by myeloid cells (e.g., killing, such as through phagocytosis) of the multispecific antigen-binding construct of this disclosure 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 microorganisms or express microbial antigens. In various embodiments, the microbial antigens are viral antigens. 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.

[0054] In at least some aspects, this disclosure provides a method for treating a subject suffering from a microbial infection, the method comprising administering an effective amount of the multispecific antigen-binding construct of this disclosure to the subject, thereby treating the subject's microbial infection. In at least some aspects, this 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 this disclosure to the subject, thereby treating the subject's cancer and / or delaying its progression. In various implementation schemes, the cancer is adenocarcinoma, bile duct (biliary tract) carcinoma, bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid, 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 implementation schemes, the method also includes administering an additional therapeutic agent to the subject for treating the cancer. In various implementation schemes, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint inhibitor.

[0055] In at least some aspects, this disclosure provides a method for treating or delaying the progression of an autoimmune or inflammatory disease in a subject, the method comprising administering to the subject an effective amount of the multispecific antigen-binding construct of this disclosure, thereby treating the subject's autoimmune or inflammatory disease and / or delaying its progression. In various embodiments, the autoimmune or inflammatory disease is atherosclerosis, obesity, inflammatory bowel disease (IBD), Lyme disease, Hashimoto's thyroiditis, autoimmune uveitis, autoimmune valvular heart 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, alopecia areata, vasculitis and temporal arteritis, graft-versus-host disease (GVHD), asthma, COPD, eosinophilia, conjunctivitis, glomerulonephritis, autoimmune nephritis, paraneoplastic autoimmune disease, cartilage inflammation, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, generalized flare-ups of juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis Inflammation, fibrotic diseases, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, systemic flare-up rheumatoid arthritis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary cholecystitis, sclerosing cholangitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse 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 abortion.

[0056] In at least some aspects, this disclosure provides a method for treating or delaying the progression of cardiovascular disease in a subject, the method comprising administering to the subject an effective amount of the multispecific antigen-binding construct of this disclosure, thereby treating the subject's cardiovascular disease and / or delaying its progression. 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, pulmonary heart disease, arrhythmia, endocarditis, myocarditis, eosinophilic myocarditis, valvular heart disease, congenital heart disease, or rheumatic heart disease.

[0057] In at least some aspects, this disclosure provides a method for treating or delaying the progression of a neurological disease in a subject, the method comprising administering to the subject an effective amount of the multispecific antigen-binding construct of this disclosure, thereby treating the subject's neurological disease and / or delaying its progression. In various embodiments, the neurological disease is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, an ophthalmic condition, 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).

[0058] In the various methods of this disclosure, the subject is a mammal, such as a human, a non-human primate, a farm animal, a domesticated animal, or a laboratory animal. In the various methods of this disclosure, the multispecific antigen-binding construct is administered orally, rectally, intravenously, intratumorally, or subcutaneously.

[0059] In at least some aspects, this disclosure provides a SIRPα-binding molecule comprising at least one heavy-chain-only variable domain (SIRPα VHH domain) comprising: a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, and 45; a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from SEQ ID NO: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In various embodiments, at least one SIRPα VHH domain comprises CDR1, CDR2, and CDR3 as follows: SEQ ID NO: 37, 46, and 61, respectively; SEQ ID NO: 38, 46, and 61, respectively; SEQ ID NO: 39, 47, and 62, respectively; SEQ ID NO: 40, 48, and 63, respectively; SEQ ID NO: 41, 49, and 64, respectively; SEQ ID NO: 37, 50, and 61, respectively; SEQ ID NO: 42, 51, and 65, respectively; SEQ ID NO: 43, 52, and 66, respectively; SEQ ID NO: 37, 53, and 67, respectively; SEQ ID NO: 44, 54, and 68, respectively; SEQ ID NO: 43, 55, and 63, respectively; SEQ ID NO: 40, 56, and 69, respectively; SEQ ID NO: 37, 57, and 70, respectively; SEQ ID NO: 40, 55, and 63, respectively; SEQ ID NO: 41, 58, and 71; 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. In various embodiments, SIRPα is human SIRPα. In various embodiments, at least one SIRPα VHH domain comprises the amino acid sequence shown in any 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 of SEQ ID NOs: 13-21 and 28-36, and binds SIRPα. In various embodiments, at least one SIRPα VHH domain comprises the amino acid sequence shown in any of SEQ ID NOs: 13-21 and 28-36.

[0060] In various embodiments, the binding of the SIRPα VHH domain to SIRPα inhibits or reduces the binding of SIRPα to differentiation cluster 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 one or more IgV domains of wild-type human SIRPα or its variants. In various embodiments, the IgV domain of one or more wild-type human SIRPα or variants thereof is selected from: (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 SEQ ID NO: 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 SEQ ID NO: 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: 105; 10. 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; and (v) 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: 2.

[0061] In various embodiments, VHH is panreactive and binds 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 to improve binding to CD47. In various embodiments, VHH binds (1) the IgV domain of wild-type allele SIRPα (optionally wild-type allele 1 and / or wild-type allele 2 SIRPα), and (2) at least one IgV domain of variant SIRPα that: (a) includes one or more amino acid substitutions in the IgV domain of wild-type SIRPα to improve binding to CD47; and / or (b) is a deglycosylated variant.

[0062] In various embodiments, the IgV domain of wild-type human SIRPα, optionally wild-type human SIRPα, comprises: (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 the variant SIRPα, optionally the variant SIRPα, contains one or more amino acid substitutions selected from the group consisting of wild-type 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 SEQ ID NO: Amino acid numbers 103 or SEQ ID NO: 104. In various embodiments, one or more amino acid substitutions include K53R, E54Q, and S66T (L66T), corresponding to amino acid numbers 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 A27I), 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 numbers 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 numbers of SEQ ID NO: 103 or SEQ ID NO: 104.

[0063] In various implementations, the variant SIRPα is FB3, FD6, FA4, or CV1. In various embodiments, the IgV domain of variant SIRPα, optionally variant SIRPα, comprises: (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; (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: 10; 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.

[0064] In at least some aspects, this disclosure provides a binding molecule comprising a SIRPα binding molecule according to this disclosure and a second binding domain for binding a second antigen. In various embodiments, the second antigen is a tumor antigen. In various embodiments, the tumor antigen is a tumor-associated antigen (TAA) selected from the group of TAAs listed in Table 2 or derived from targets listed in Table 2. In various embodiments, 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). In various embodiments, the binding domain for binding the second antigen is an antibody or an antigen-binding fragment.

[0065] In at least some aspects, this disclosure provides nucleic acids encoding SIRPα-binding molecules according to this disclosure. In at least some aspects, this disclosure provides expression vectors comprising the nucleic acids of this disclosure. In at least some aspects, this disclosure provides cells comprising the expression vectors of this disclosure. In at least some aspects, this disclosure provides a method for producing SIRPα-binding molecules, the method comprising culturing cells of this disclosure or populations of such cells under conditions favorable to expression of the SIRPα-binding molecules from the expression vectors. In various embodiments, the method for producing SIRPα-binding molecules as provided herein further includes isolating the SIRPα-binding molecules from cells or cell populations, or from a culture medium in which the cells or cell populations are cultured. In at least some aspects, this disclosure provides a pharmaceutical composition comprising the SIRPα-binding molecule of this disclosure and a pharmaceutically acceptable carrier or excipient.

[0066] In at least some aspects, this disclosure provides a method for treating a subject with cancer or delaying its progression, the method comprising administering to the subject an effective amount of the disclosed SIRPα-binding molecule, binding molecule, or pharmaceutical composition, thereby treating the subject's cancer and / or delaying its progression. In various embodiments, the cancer is adenocarcinoma, bile duct (biliary tract) cancer, bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid tumor, 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 to the subject an additional therapeutic agent for treating the cancer. In various implementation schemes, the additional treatment agents are chemotherapeutic agents or checkpoint inhibitors. Attached Figure Description

[0067] Figure 1A Drawings of six anti-EGFR antibodies are shown. The constructs include: nexituzumab (11F8) hIgG1 (C-1), nexituzumab (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) contains G44. H C / G100 LC scFv stabilization mutation. EEF = Enhanced effector function (via G236A / S239D / I332E mutations in FC, indicated by dots). Figure 1B Results of flow cytometry assays are shown to assess EGFR cell binding of anti-EGFR antibodies on A431 (high EGFR) and MCF7 (low EGFR) cells. Results are presented as mean fluorescence intensity (MFI).

[0068] Figure 2A Drawings show four anti-EGFR-SIRPα fusion constructs containing the EGFR antibody nexituzumab (11F8) hIgG1 EEF or 11F8 scFv IgG1 EEF and domain 1 (SIRPα-D1) of SIRPα. The position of SIRPα varies in the constructs. For the 11F8 scFv IgG1 EEF-SIRPα fusion construct, SIRPα-D1 is fused to the C-terminus of the Fc (C-7). For the nexituzumab (11F8) hIgG1 EEF-SIRPα fusion construct, SIRPα-D1 is fused to the C-terminus of the heavy chain (fusion 1, C-8), to the C-terminus of the light chain (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 the C-terminal light chain fusion construct (C-9) contain G44. H C / G100 L CscFv stabilization mutation. EEF = Enhanced effector function (via G236A / S239D / I332E mutations in FC, indicated by dots).

[0069] Figure 2B Results of flow cytometry assays are shown to evaluate CD47 cell binding of the anti-EGFR-SIRPα fusion construct on MCF7 WT cells (CD47+ / EGFR-) and MCF7 CD47 knockout cells (KO; CD47- / EGFR-). Results are shown as mean fluorescence intensity (MFI). Resistuzumab hIgG1 EEF (C-2) and SIRPα-free 11F8 scFv hIgG1 EEF (C-6) were used as negative controls for CD47 binding.

[0070] Figure 3A A plot showing the anti-EGFR double complementation site VHH hIgG1 EEF SIRPα fusion construct (C-64) and the control construct without SIRPα fusion (C-63). EEF = enhanced effector function (indicated by the G236A / S239D / I332E mutation in FC, represented by dots).

[0071] Figure 3B Results of flow cytometry assays are shown to evaluate CD47 cell binding of the anti-EGFR double complement VHH hIgG1 EEF SIRPα fusion (C-64) on MCF7 WT cells (CD47+ / EGFR-) and MCF7 CD47 knockout cells (KO; CD47- / EGFR-). Results are shown as mean fluorescence intensity (MFI). Resistuzumab hIgG1 EEF (C-2) and the SIRPα-free anti-EGFR double complement VHH hIgG1 EEF construct (C-63) were used as negative controls for CD47 binding.

[0072] Figure 3C Results of flow cytometry assays are shown to evaluate EGFR cell binding of the anti-EGFR double complement VHH hIgG1 EEF SIRPα fusion (C-64) on A431 WT (high EGFR / CD47+) and A431 CD47 KO (high EGFR / CD47-) cells. Results are shown as mean fluorescence intensity (MFI). Resistuzumab hIgG1 EEF (C-2) and the SIRPα-free anti-EGFR double complement VHH hIgG1 EEF construct (C-63) were used as positive controls for EGFR binding.

[0073] Figure 4A A drawing of an anti-EGFR-anti-CD47 fusion construct containing the EGFR antibody nexituzumab (11F8), hIgG1 EEF, and the anti-CD47 VHH antibody is shown. The position of the anti-CD47 VHH antibody varies in the constructs: the anti-CD47 VHH antibody is 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) contains G44. H C / G100 L C scFv stabilization mutation. EEF = Enhanced effector function (via G236A / S239D / I332E mutations in FC, indicated by dots).

[0074] Figure 4B Results of flow cytometry assays are shown to evaluate CD47 cell binding of the nexituzumab-anti-CD47 VHH fusion in MCF7 WT cells (CD47+ / EGFR-) and MCF7 CD47 knockout cells (KO; CD47- / EGFR-). Results are shown as mean fluorescence intensity (MFI). Nexituzumab hIgG1 EEF (C-2) was used as a negative control for CD47 binding.

[0075] Figure 5A An exemplary workflow for phagocytic screening of a multispecific antigen construct is shown. THP-1 monocytes stably expressing the nuclear restriction tag GFP2 were used as effector cells, while A431 human epidermal-like cancer cells labeled with pHrodo Orange were used as target cells. Cells were co-incubated with the multispecific antigen construct and imaged every 45 minutes using brightfield, green, and orange channels for 12 hours. Phagocytized cells were defined as green + high-intensity orange overlap because pHrodo fluoresces only at low pH (after phagocytosis).

[0076] Figure 5B The amount of phagocytosis is shown after adding each fusion construct (C-1-C-15 and C-64-C-64) at a concentration of 50 nM. The results are shown as the area under the curve (AUC).

[0077] Figure 5C The amount of phagocytosis following the addition of 0.5, 5, and 50 nM of the nexituzumab (11F8) hIgG1 EEF SIRPα fusion constructs (C-8, C-9, and C-10) is shown. Results are presented as area under the curve (AUC). The nexituzumab (11F8) hIgG1 (C1) and nexituzumab (11F8) hIgG1 EEF (C2) constructs were used as non-SIRPα controls. A control lacking the anti-EGFR domain was also used, alone or in combination with nexituzumab (11F8) hIgG1 EEF (C2, anti-EGFR only), in which SIRPα was fused to the C-terminus of the homodimeric Fc IgG1 EFF peptide (C-14, CD47 blocker only).

[0078] Figure 5D The amount of phagocytosis is shown after adding a CD47 blocker-only (C14, Fc IgG1 EFF-SIRPα) construct, a tumor-targeting construct (nexituzumab (11F8) hIgG1 EEF; C2), a combination of CD47 blocker (C14) and anti-EGFR (C2) constructs, or as a multispecific fusion construct (nexituzumab (11F8) hIgG1 EEF SIRPα fusion 1; C-8) at a concentration of 0.5 nM. Results are shown as fold changes in phagocytosis. An asterisk indicates P < 0.0001, as determined by two-way ANOVA combined with Tukey's multiple comparison test.

[0079] Figure 6ADrawings of CD47-only (anti-EGFR-free) constructs are shown, in which SIRPα D1 is fused to the C-terminus of a homodimeric FcIgG1 EFF peptide (SIRPα D1 Fc fusion 1, C-14) or to its N-terminus (SIRPα D1 Fc fusion 2, C-15). Constructs in which the CD47 extracellular domain is used as a “masking domain” to block the interaction between SIRPα-D1 and cell surface CD47 are also depicted. The CD47 extracellular domain and SIRPα-D1 are each linked to one of the polypeptide chains of heterodimeric Fc with a club-and-mortise mutation, with this linkage occurring at the N-terminus of each Fc polypeptide (KiH Fc EEF CD47 and SIRPα fusion 1, C-16M) or at its C-terminus (KiH Fc EEF CD47 and SIRPα fusion 2, C-17M). The masking domain is connected to the Fc region via a cuttable connector to conditionally control the interaction between the CD47 extracellular domain and SIRPα D1.

[0080] Figure 6B Results of flow cytometry assays are shown to evaluate the binding of SIRPαD1-Fc fusions (C14, C15, C16M, and C17M) with or without masking domains to CD47 cells in MCF7 WT (CD47+ / EGFR-), MCF7 KO (CD47- / EGFR+), A431 WT (CD47+ / EGFR+), and A431 KO (CD47+ / EGFR+) cells. Results are shown as mean fluorescence intensity (MFI).

[0081] Figure 7A Drawings show three forms of the anti-EGFR-SIRPα-maskant fusion construct. These forms include: nexituzumab (11F8) hIgG1 EEF:SIRPα-WT:maskant fusion (Form #1); nexituzumab (11F8) hIgG1 EEF:SIRPα-CV1:maskant fusion (Form #2); and the asymmetric variant KiH nexituzumab (11F8) hIgG1 EEF, in which the acetabular arm is fused with SIRPα-CV1 and the club arm is fused with the maskant (Form #3).

[0082] Figure 7BResults of flow cytometry assays are shown to evaluate CD47 cell binding of the anti-EGFR-SIRPα-masker fusion construct on MCF7 WT (CD47+ / EGFR-) and MCF7 KO (CD47- / EGFR+) cells. The fusion construct was assayed with or without digestion with matrix protease at concentrations ranging from 100 nM to 0.6 pM. Exemplary results for the constructs are shown: no mask (C-50), masked + protease (C-55M digested), masked (C-55M undigested), and masked with an uncleavable linker (C61-M). Results are shown as median fluorescence intensity (MFI) plotted relative to the concentration of the fusion construct. Detailed Implementation

[0083] This disclosure relates to compositions and methods for treating diseases (e.g., cancer). In particular, this disclosure provides compositions that selectively direct myeloid cell activity to target cells (e.g., direct and / or indirect killing of target cells by myeloid cells), such as through selective activation under specific target conditions, such as activation of the tumor microenvironment. Among the provided embodiments is a multispecific binding molecule platform that is selectively activated in tumors to promote macrophage killing of tumor cells. This platform includes various configurations that are combined for targeting tumors, pre-set with masking domains to achieve conditional activation, thereby blocking antiphagocytic protein activity in a tumor-selective manner and promoting macrophage-dependent antibody-dependent cytotoxicity (ADCC) and phagocytosis (ADCP), thereby promoting tumor cell killing. In some embodiments, the therapeutic binders covered by this disclosure can selectively direct the elimination of target cells via antibody-dependent cytotoxicity (ADCC). In some embodiments, the therapeutic binders covered by this disclosure can selectively direct the elimination of target cells via antibody-dependent cytotoxicity (ADCC). In some implementations, the therapeutic binders covered by this disclosure can selectively guide the elimination of target cells through direct killing.

[0084] There is a need for improved therapeutic agents that can promote the potential cytotoxic activity of myeloid cells, including macrophages. Macrophages account for 50% of all cells in solid tumors (Mellman et al., Immunity (2023); Cheng et al. J. Hematology Oncology(2023)). However, current therapies have not effectively utilized macrophages to directly kill tumors. For example, the immunosuppressive tumor microenvironment created by macrophages is a major obstacle to the success of immuno-oncology. Although macrophages are capable of engulfing and killing tumor cells, they are often inactive and immunosuppressive in the tumor microenvironment. For example, many tumor cells and cells in the tumor microenvironment express CD47 and inhibit the phagocytic activity of macrophages. Therefore, the therapeutic potential of macrophages has not been fully realized, including the potential for macrophages to promote rapid innate immune-driven tumor reduction and to bridge innate and adaptive immunity to achieve a durable clinical response. Therefore, there is an urgent need to unlock macrophages to directly kill tumors, including as part of a coordinated immune attack, thereby enabling a durable response.

[0085] In some embodiments, the therapeutic binders covered by this disclosure may comprise: (1) at least one binding domain that binds an antiphagocytic protein (APP) (e.g., expressed on myeloid cells or on cells targeted to regulate (e.g., direct and / or indirect killing)) (APP binding domain), (2) at least one binding domain that conditionally inhibits the interaction of the antiAPP binding domain with its target (masking domain), and (3) a proteolytically cleavable linker whose localization allows its cleavage to release the inhibition of APP binding by the masking domain, thereby enabling activatable activity. The therapeutic binders covered by this disclosure may comprise one or more additional elements, such as one or more elements that promote and / or activate myeloid cells, and / or at least one binding domain that binds a target cell antigen (target cell binding domain). For example, the provided binders are multispecific binders that also include an immunoglobulin Fc region for activating FcγR. In some embodiments, the immunoglobulin Fc region contains an Fc-enhancing mutation, which is also known to enhance macrophage activity to promote macrophage-dependent ADCC / ADCP. Fc enhancing mutations are mutations that increase affinity for FcγRIIIa (e.g., S239D and I332E) and mutations that increase affinity for FcγRIIa relative to the inhibitory receptor FcγRIIb (e.g., G236A). For example, the Fc enhancing triple mutants S239D, I332E, and G236A can increase the binding ratio of RIIa to RIIb while maintaining enhanced affinity for FcγRIIIa. This article also describes these elements, therapeutic binders including one or more of such elements, and their uses, for example, in the treatment of cancer.

[0086] In the provided embodiment of the binder, the APP is CD47, and the binder includes at least one binding domain to bind CD47, thereby blocking the inhibition of macrophages and other myeloid cells. CD47 blockade can induce long-term anti-tumor immunity and can also bridge innate and adaptive immune systems. Furthermore, combining CD47 blockade with a binder that allows FcγR interaction can promote myeloid-directed anti-tumor responses, including responses to distant tumors. Additionally, using a masking domain that can be released in the tumor, the provided binder is conditionally activated to conditionally control the specific binding of APP in the tumor microenvironment, rather than in tumor tissue, thereby maximizing selective activation in the tumor to promote macrophage-specific killing of tumor cells, rather than normal cells. For example, in the provided embodiment, tumor selectivity can be achieved by activation of a protease-mediated, proteolytically cleavable linker that connects the masking domain to the APP binding domain, thereby selectively activating the provided binder in the tumor. This addresses some shortcomings of existing CD47 blockade therapies, as CD47 is ubiquitous in both normal and tumor cells. Extratumor binding of CD47 can lead to toxicity and adverse pharmacology, including anemia, thrombocytopenia, and tissue accumulation (reduced availability), while expected tumor-specific targeted binding results in antitumor activity and promotes ADCC / ADCP, thereby enhancing tumor cell killing. The provided approach also offers the flexibility to modulate binding by using the SIRPα binding domain as the binding domain for CD47 apps, including wild-type SIRPα and various engineered variants with intermediate or high affinity. The provided approach offers unique therapeutic agents that allow for enhanced Fc function and CD47 blockade while avoiding other compromises such as Fc silencing or the use of low-affinity CD47 blockers.

[0087] This disclosure also relates to functional equivalents of the following: APP-binding domains (i.e., APP-binding domain devices), masking domains (i.e., masking domain devices), proteolytically cleavable adapters (i.e., proteolytically cleavable adapter devices), myeloid cell promoting and / or activating elements (i.e., myeloid cell promoting devices and / or myeloid cell activating devices), and / or target cell binding domains (i.e., target cell binding domain devices). Functional equivalents of each corresponding element will not differ materially from the corresponding elements herein, as they perform substantially the same functions in substantially the same manner to achieve substantially the same results. Functional equivalents of each corresponding element can be prepared and / or screened by well-known methods, such as assays for analyzing binding, affinity, phagocytosis (e.g., ADCP), cell killing (e.g., ADCC or CDC), cytokine release, immune cell activation, immune cell proliferation, and / or immune cell migration, and representative examples of their functions, elements, and / or equivalents are further described herein.

[0088] This disclosure is based, at least in part, on the understanding that cells associated with certain diseases, symptoms, or conditions (these terms are used interchangeably herein) (such as cancer) can evade phagocytosis through aberrant and / or increased expression of APP, leading to a pathological state. Increased expression of APP (e.g., CD47) inhibits phagocytosis and is associated with poor prognosis. The binding agents provided herein conditionally (activatably) direct myeloid cell activity to such cells, thereby treating the disease.

[0089] All publications (including patent documents, scientific literature, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication were incorporated individually by reference. Where the definitions listed herein contradict or otherwise are inconsistent with those shown in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions shown herein shall prevail over those incorporated herein by reference.

[0090] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0091] I. Definition One, a kind, the said / the, or As used herein, “a,” “an,” and “the” refer to one or more of the grammatical objects of the article (i.e., at least one or at least one). For example, “(a) element” discloses an embodiment with only one element and an embodiment including more than one element. As used herein, the terms “or” and “and / or,” as conjunctions in a list of at least two elements, cover and disclose embodiments in which the listed elements are included together or in any combination in an alternative.

[0092] about: In some embodiments, the term "about" covers a value within any range (e.g., plus or minus 2% to 6%) of or between 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (inclusive) of the measured value. In some embodiments, the term "about" refers to the inherent variation in error in the method, determination, or measured value, such as variations that exist between experiments.

[0093] Affinity:As used herein, “affinity” refers to the strength of the sum of non-covalent interactions between a particular binder (e.g., an antigen binder) and / or its binding site with its binding target (e.g., an antigen). Unless otherwise stated, as used herein, “binding affinity” refers to a 1:1 interaction between a binder and its binding target (e.g., an antibody and its antigen target). Those skilled in the art will understand that changes in affinity can be described by comparison with a reference (e.g., an increase or decrease relative to a reference) or can be described numerically. Affinity can be measured and / or represented in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (K0). D ) and / or equilibrium association constant (K A K D It is k off / k on The merchant, and K A It is k on / k off The quotient of which k on This refers to, for example, the association rate constant between an antibody and an antigen, and k off This refers to, for example, the dissociation of antibodies and antigens. on and k off This can be achieved through techniques known to those skilled in the art (such as BIACORE) ® It can be determined by either KinExA or KinExA.

[0094] Agent As used herein, the term "agent" may refer to any chemical entity, including but not limited to atoms, molecules, compounds, amino acids, polypeptides, nucleotides, nucleic acids, proteins, protein complexes, liquids, solutions, sugars, polysaccharides, lipids, or combinations or complexes thereof.

[0095] Antibody As used herein, the term "antibody" refers to a polypeptide comprising one or more canonical 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 pharmaceutical agents comprising them. Antibodies may be naturally occurring immunoglobulins (e.g., generated by an organism that reacts with an antigen). Synthetic, non-natural, or engineered antibodies may be produced through recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those skilled in the art.

[0096] As is well known in the art, a typical human immunoglobulin is a tetramer of approximately 150 kDa, comprising two identical heavy (H) chain polypeptides (approximately 50 kDa each) and two identical light (L) chain polypeptides (approximately 25 kDa each), which associate with each other to form a structure commonly referred to as a “Y-shape.” Typically, each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain comprises three CH domains: CH1, CH2, and CH3. Short regions called “switches” connect the heavy chain variable and constant domains. “Hinges” connect the CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain contains a light chain variable domain (VL) and a light chain constant domain (CL), separated from each other by another “switch.” Each variable domain contains three hypervariable loops (CDR1, CDR2, and CDR3) called “complementarity-determining regions” and four relatively invariant “framework” regions (FR1, FR2, FR3, and FR4). In each VH and VL, three CDRs and four FRs are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and / or light chains are generally understood to provide binding portions that can interact with antigens. Constant domains mediate the binding of antibodies to various immune system cells (e.g., effector cells and / or cells mediating cytotoxicity), receptors, and complement system components. The heavy and light chains are linked to each other by a single disulfide bond, and two additional disulfide bonds link the heavy chain hinge regions together, causing the dimers to link together and form a tetramer. When innate immunoglobulins fold, the FR regions form β-sheets that provide a structural framework for the domains, and the CDR loop regions from the heavy and light chains aggregate in three-dimensional space, forming a single hypervariable antigen-binding site located at the tip of the Y structure.

[0097] 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" can include (unless otherwise stated or clear from the context) any construct or form known in the art that utilizes the structural and / or functional characteristics of an antibody, including but not limited to: intracellular antibodies, domain antibodies, antibody mimics, Zybody®, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, isolated CDRs or groups thereof, single-chain antibodies, single-chain Fv (scFv), disulfide-linked Fv (sdFv), peptide-Fc fusions, single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof), camel antibodies, camel-derived antibodies, masking antibodies (e.g., Probody®), affybody, anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), single-chain or tandem biantibodies (TandAb®), VHH, Anticalin®, Nanobody® small antibodies, BiTE®, ankyrin repeat protein 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.

[0098] Single domain antibodiesAs used herein, the term "single-domain antibody" can be interchangeably referred to as "VHH domain" or "heavy-chain-only antibody variable domain," referring to a single-chain antigen-binding domain capable of binding an antigen or epitope independently of the second light-chain variable domain. A single-domain antibody VHH differs from the heavy-chain variable domain (referred to herein as the "VH domain" or "VH region") present in conventional four-chain antibodies and the light-chain variable domain (referred herein as the "VL domain" or "VL domain") present in conventional four-chain antibodies. VHH domains can be human domains, but also include single-domain domains from other species, such as rodent, nurse shark, and camelid VHH domains. Camelid VHHs are immunoglobulin single-variable-domain polypeptides derived from species including camels, llamas, alpacas, dromedary camels, and guanacos, which produce naturally occurring heavy-chain antibodies lacking the light chain. Such VHH domains can be humanized using standard techniques available in the art and are considered "single-domain antibodies." VHHs include 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, including humanoid VHH domains, such as those in which 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 a camel VHH (see, for example, PCT Publication WO2021 / 178263).

[0099] A "humanized" antibody is an antibody (e.g., a VHH) that comprises amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, all or substantially all of the CDRs of a humanized VHH correspond to the CDRs of a non-human VHH, and all or substantially all of the FRs correspond to the FRs of a human antibody. Optionally, a humanized antibody may contain at least a portion of the antibody constant region derived from a human antibody.

[0100] "Affinity-maturated" antibodies (such as VHH) have one or more modifications in one or more CDRs that result in enhanced affinity for the antigen compared to the corresponding parental albumin-binding molecule. The affinity-maturated albumin-binding molecule of the present invention can be prepared by methods known in the art, for example, as described in KS Johnson and RE Hawkins, "Affinitymaturation of antibodies using phage display", Oxford University Press, 1996.

[0101] In some embodiments, the antibody comprises one or more structural elements that are 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 (“conjugated”) antibody (e.g., an antibody comprising a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, wherein the polypeptide is covalently linked to one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, the antibody sequence element is humanized, primate-derived, chimeric, etc., as known in the art.

[0102] Antibodies including the heavy chain constant domain, based on the amino acid sequence of the heavy chain constant domain (e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (µ)), can be, but are not limited to, any known class of antibodies, including but not limited to IgA, secretory IgA, IgG, IgE, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an Ab class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant domain gene. As used herein, based on the amino acid sequence of the light chain constant domain, the "light chain" can be of different types, such as kappa (κ) or lambda (λ). In some embodiments, the antibody has a constant region sequence characteristic of mouse, rabbit, primate, or human immunoglobulins. Naturally occurring immunoglobulins are glycosylated, typically glycosylated at the CH2 domain. As is known in the art, the affinity and / or other binding properties of the Fc domain (which is interchangeably referred to as the "Fc region") for the Fc receptor can be modulated by glycosylation or other modifications. In some embodiments, the antibody may lack the covalent modifications (e.g., attachment glycans) that would be present in naturally occurring antibodies. In some embodiments, antibodies generated and / or utilized according to this disclosure include a glycosylated Fc domain, including a modified or engineered Fc domain.

[0103] Antibody-dependent cell-mediated cytotoxicity (ADCC) The term refers to a form of cytotoxicity in which a secreted antibody binds to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to and subsequently kill target cells carrying antigens. To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. As is well known in the art, the Fc portion can be engineered to achieve a desired interaction with the Fc receptor or to lack such an interaction.

[0104] antibody fragments As used herein, "antibody fragment" means an antibody fragment or antibody agent as described herein, and generally refers to a portion comprising an antigen-binding portion or its variable region. Antibody fragments can be generated in any manner. For example, in some embodiments, antibody fragments can be generated enzymatically or chemically by fragmentation of a complete antibody or antibody agent. Alternatively, in some embodiments, antibody fragments can be generated recombinantly (i.e., by expressing an engineered nucleic acid sequence). In some embodiments, antibody fragments can be generated entirely or partially synthetically. In some embodiments, the length of the antibody fragment (particularly an antigen-binding antibody fragment) can be 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 at least about 200 amino acids.

[0105] Antigen-binding domain:The term "antigen-binding domain" or "binding portion" refers to a portion of a binding molecule (such as an immunoglobulin molecule) involved in antigen binding. For conventional four-chain antibodies or Fab fragments, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments), or biantibodies or other antibody fragments derived from conventional four-chain antibodies, the antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") region of the heavy ("H") and light ("L") chains. In these cases, binding to the corresponding epitope of the antigen occurs via a pair of (associated) immunoglobulin domains (such as the light and heavy chain variable domains), i.e., via the VH-VL pair of immunoglobulin domains, which jointly bind the epitope of the corresponding antigen. Three highly divergent segments (called "hypervariates") within the V region of the heavy and light chains are located between more conserved flanking segments called "frame regions" or "FRs." Therefore, the term "FR" refers to the amino acid sequence between and near the hypervariates naturally present in immunoglobulins. In antibody molecules, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface that binds the antigen, and the three hypervariable regions of each of the heavy and light chains are called “complementarity-determining regions” or “CDRs”. The amino acid assignment of each domain conforms to the definitions in the following literature: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia and 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 includes a heavy chain variable domain, which comprises the three CDRs of the heavy chain variable domain. The VHH domain can specifically bind epitopes in the absence of additional antigen-binding domains (unlike the VH or VL domains in conventional 4-chain antibodies, where epitopes are typically recognized by both the VL and VH domains). Many proteins also include immunoglobulin domains, called immunoglobulin-like (Ig-like) domains, which are protein regions homologous to the V or C domains of immunoglobulin proteins responsible for binding antigens in immunoglobulins. These immunoglobulin domains in proteins of the immunoglobulin superfamily are classified as IgV or IgC domains and can act as antigen-binding domains involved in antigen binding. Related to:The two events or entities are “associated” with each other, as used herein, if the presence, level, and / or form of one event or entity is related to the presence, level, and / or form of another event or entity. For example, if the presence, level, and / or form of a particular entity (e.g., polypeptide, genetic trait, metabolite, microorganism, etc.) is related to the incidence and / or susceptibility to a particular disease, condition, or symptom (e.g., within a relevant population), then that particular entity is considered to be associated with that disease, condition, or symptom. In some embodiments, two or more entities are physically “associated” with each other if they interact directly or indirectly such that they are physically close to each other and / or remain physically close. In some embodiments, two or more physically associated entities are covalently linked to each other; in some embodiments, two or more physically associated entities are not covalently linked to each other but are non-covalently associated, for example, by means of hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, or combinations thereof.

[0106] Combination As used herein, the term "binding" refers to a non-covalent association between two or more agents. "Direct" binding involves physical contact between agents; indirect binding involves physical interaction through physical contact with one or more intermediate agents. Binding between two or more agents can occur and / or be evaluated in any of a variety of situations, including where the interacting agents are studied individually or in more complex systems (e.g., when covalently or otherwise associated with a carrier and / or in biological systems or cells).

[0107] 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 to or has a high affinity for that partner. The strength or affinity of an immune binding interaction can be expressed by the dissociation constant (K0) of the interaction. D Let K be the smallest value of K. DThe greater the affinity, the better. The immunobinding properties of a selected peptide can be quantified using methods well known in the art. One such method requires measuring the rates of antigen binding site / antigen complex formation and dissociation, where these rates depend on the concentration of the complex conjugate, the affinity of the interaction, and geometric parameters that have an equal effect on both rates. Thus, both the “binding rate constant” (Kon) and the “dissociation rate constant” (Koff) can be determined by calculating the concentration and the actual association and dissociation rates (see Nature 361:186-87 (1993)). The ratio of Koff / Kon allows for the cancellation of all affinity-independent parameters, and this ratio is equal to the dissociation constant Kd (generally see Davies et al. (1990) Annual Rev Biochem 59:439-473). When the binding constant (Kd) is ≤1µM, for example, ≤100nM in some embodiments, ≤10nM in some embodiments, and ≤100pM to about 1pM in some embodiments, the antibody of this disclosure is considered to specifically bind to an antigen (e.g., EGFR), as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0108] A protein (such as a protein, immunoglobulin, antibody, or immunoglobulin single variable domain) that can “bind” or “specifically bind” to an epitope, antigen, or protein, and that has “affinity” and / or “specificity” to an epitope, antigen, or protein, is referred to as “targeting” or “directly targeting” the epitope, antigen, or protein, or as a “binding” molecule with respect to that epitope, antigen, or protein.

[0109] cancer: As used herein, the term "cancer" refers to a disease, condition, or symptom in which cells exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in an abnormally elevated rate of proliferation and / or an abnormal growth phenotype, characterized by a significant loss of control over cell proliferation. In some embodiments, cancer may include one or more tumors. In some embodiments, cancer may be or include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, cancer may be or include solid tumors. In some embodiments, cancer may be or include hematologic malignancies.

[0110] ChemotherapyAs used herein, the term "chemotherapeutic agent" is consistent with its use in the art and refers to one or more agents known to treat or aid in the treatment of cancer, or having properties known to treat or aid in the treatment of cancer. In particular, chemotherapeutic agents include apoptosis-inducing agents, cell inhibitors, and / or cytotoxic agents. In some embodiments, chemotherapeutic agents may be or include: alkylating agents, anthracyclines, cytoskeleton disruptors (e.g., microtubule-targeting fractions, such as taxanes, maytansine, and analogues thereof), epothilone, histone deacetylase inhibitors (HDAC), 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 having related antiproliferative activity. In some specific embodiments, the chemotherapeutic agents may be or include: actinomycin, all-trans retinoic acid, oliquistatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, deoxyfluorouridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogues (e.g., DM1), nitrogen mustard, mercaptopurine, methotrexate, mitoxantrone, maytansine compounds, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, pentorubicin, vincristine, vinblastine, vinorelbine, vinorelbine, or combinations thereof. In some implementations, chemotherapeutic agents can be used in the case of antibody-drug conjugates.In some embodiments, the chemotherapeutic agent is a chemotherapeutic agent present in a selection of the following antibody-drug conjugates: 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, h LL1-Pro-2-P-Dox, P4 / D10-Doxorubicin, Gelatinumab Ozomicin, Bentuximab Vidocin, Trastuzumab Metancin, Intuzumab Ozomicin, Gabatumumab Vidocin, 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 ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vortexuzumab malvodotin, and lovotuzumab motansin. In some embodiments, the chemotherapeutic agent may be or include: farnesylthiosalicylic acid (FTS), 4-(4-chloro-2-methylphenoxy)-N-hydroxybutyramide (CMH), estradiol (E2), tetramethoxystilbene (TMS), δ-tocotrienol, salinomycin, or curcumin.

[0111] Complement-dependent cytotoxicity (CDC) The term refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is triggered by the binding of the first component of the complement system to an antibody that binds to its homologous antigen. To assess complement activation, a CDC assay can be performed, for example, as described by Gazzano-Santoro et al. (1997).

[0112] Domain:As used herein, the term "domain" refers to a segment or portion of an entity. In some embodiments, a "domain" is associated with a specific structural and / or functional characteristic of an entity such that when the domain is physically separated from the remainder of its parent entity, the domain substantially or completely retains the specific structural and / or functional characteristic. Alternatively or additionally, a domain may be or comprise a portion of an entity that, when separated from the (parent) entity and attached to a different (receiving) entity, substantially retains and / or imparts to the receiving entity one or more of the structural and / or functional characteristics that characterize it in the parent entity. In some embodiments, a domain is a segment or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a segment of a polypeptide; in some such embodiments, the domain is characterized by specific structural elements (e.g., specific amino acid sequences or sequence motifs, α-helix characteristics, β-sheet characteristics, coil-coil characteristics, random coil characteristics, etc.) and / or specific functional characteristics (e.g., binding activity, enzymatic activity, folding activity, signal transduction activity, etc.). In some implementations, a domain is or includes characteristic portions or characteristic sequence elements.

[0113] Environment or microenvironment: The terms "environment" and "microenvironment" generally refer to a local area or characteristic of a target tissue region, and may, for example, refer to the "tumor microenvironment." The term "tumor microenvironment" or "TME" refers to the surrounding microenvironment that constantly interacts with tumor cells, facilitating cross-communication between tumor cells and their environment. The tumor microenvironment can 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 can include tumor cells or malignant cells that are aided 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 cells and myeloid cells, that can stimulate or inhibit anti-tumor immune responses, and stromal cells, such as tumor-associated fibroblasts and endothelial cells, that contribute to the structural integrity of the tumor. Stromal cells can include cells that form tumor-associated blood vessels, such as endothelial cells and pericytes, which contribute to structural integrity (fibroblasts), 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. Stromal cells make up the bulk of tumor cells, while macrophages are the dominant cell type in solid tumors.

[0114] Excerpt:As used herein, a “fragment” refers to a structure that is or includes a discrete portion of a reference agent (sometimes referred to as a “parent” agent). In some embodiments, the fragment lacks one or more portions present in the reference agent. In some embodiments, the fragment is or includes one or more portions present in the reference agent. In some embodiments, the reference agent is a molecule, such as a small molecule or other chemical entity. In some embodiments, the fragment of the molecule is or includes 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 present in the reference molecule.

[0115] In some embodiments, the reference agent is a polymer, such as a polynucleotide or polypeptide. In some embodiments, the fragment of the polymer is or includes 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, 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) of the reference polymer. In some embodiments, the polymer fragment is or comprises 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 monomer units (e.g., residues) present in the reference polymer. The fragment of the reference polymer is not necessarily identical to the corresponding portion of the reference polymer. For example, the fragment of the reference polymer may be a polymer having a residue sequence having 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 identity with the reference polymer. The fragment may or may not be generated by physical fragmentation of the reference agent. In some cases, the fragment is generated by physical fragmentation of the reference agent. In some cases, fragments are not generated through the physical breakdown of the reference agent, but can be generated, for example, through... From the beginning It can be produced by synthesis or other means.

[0116] improve , Increase / improve , inhibition , reduce or reduce As used herein, the terms “improve,” “increase / enhance,” “suppress,” “reduce,” and “reduc,” and their grammatical equivalents, indicate qualitative or quantitative differences from a reference.

[0117] Inhibit or downregulate: The terms “inhibition” or “downregulation” include, for example, a reduction, restriction, or obstruction of a particular action, function, or interaction. In some embodiments, cancer is “inhibited” if at least one symptom of cancer is relieved, terminated, slowed, or prevented. As used herein, cancer is also “inhibited” if recurrence or metastasis of cancer is reduced, slowed, delayed, or prevented. 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 a wild-type state). Such inhibition or deficiency can be induced (e.g., by administration of an agent at a specific time and / or location) or can be constitutive (e.g., by a heritable mutation). Such inhibition or deficiency can also be partial or complete (e.g., substantially no measurable activity compared to a reference state, such as a wild-type state). In some embodiments, substantially complete inhibition or deficiency is referred to as “blockade.” In one embodiment, the term refers to reducing the level of a given output or parameter to an amount (e.g., background staining, biomarker signaling, biomarker immunosuppressive function, etc.) that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more lower than that in a corresponding control. A reduction in the level of a given output or parameter does not need to (although may) imply the absolute absence of the output or parameter. This disclosure does not require, and is not limited to, methods for completely eliminating the output or parameter. Methods well known in the art can be used to determine a given output or parameter, including but not limited to immunohistochemistry, molecular biology, cell biology, clinical and biochemical assays, as discussed herein. The terms “promote” and “upregulate” have opposite meanings.

[0118] Connector:As used herein, "linker" refers to the portion of a multi-element agent that connects different elements to each other. For example, those skilled in the art will understand that peptides whose structure comprises two or more functional or tissue domains typically include a segment of amino acids located between such domains and connecting them to each other. In some embodiments, the peptide containing the linker element has an overall structure of the general form S1-L-S2, wherein S1 and S2 may be the same or different, and represent two domains associated with each other by the linker. In some embodiments, the length of the peptide linker is 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 amino acids. In some implementations, the linker is characterized by its tendency to provide flexibility to the peptide rather than employing a rigid three-dimensional structure. When the peptide is engineered (e.g., a fusion peptide), a variety of different linking elements known in the art can be appropriately used (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2: 1 121-1123).

[0119] adjust: The term “regulation” and its grammatical equivalents refer to and encompass either or both of increasing / raising and decreasing / lowering.

[0120] Operable connection As used herein, "operably linked" means the association of at least a first element and a second element such that the constituent elements are in a relationship that allows them to function in the intended manner. For example, a nucleic acid sequence or amino acid sequence is operably linked to another sequence if, for example, it modifies the expression, structure, or activity of the linked sequence in the intended manner. In many cases, two nucleic acid sequences are operably linked if they contribute to the expression, structure, or activity of a gene or its encoded polypeptide. In many cases, two amino acid sequences are operably linked if they are expressed as a single polypeptide.

[0121] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” when applied to one or more or all components used to formulate compositions as disclosed herein means that each component must be compatible with the other components of the composition and harmless to its recipient.

[0122] Pharmaceutically acceptable carriers:As used herein, the term “pharmaceuticalally acceptable carrier” refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that facilitates the formulation of an agent (e.g., a pharmaceutical preparation), alters the bioavailability of the agent, or facilitates the transport of the agent from one organ or part of a subject to another organ or part of the subject. Some examples of materials that can be used 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; 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 glycerol, 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; ethanol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic and compatible substances used in pharmaceutical formulations.

[0123] Pharmaceutical compositions or formulations 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.

[0124] Reference point: As used herein, a “reference object” refers to a standard or control relative to which comparison is made. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual or a population thereof, or a measure or characteristic representing it, is compared with a reference object, agent, sample, sequence, subject, animal, or individual or a population thereof, or a measure or characteristic representing it. In some embodiments, the reference object is a measurement. In some embodiments, the reference object is an established standard or expected value. In some embodiments, the reference object is a historical reference. The reference object can be quantitative or qualitative. Generally, as those skilled in the art will understand, the reference object and the value compared to it represent an assessment performed under comparable conditions. Those skilled in the art will understand when sufficient similarity exists to justify dependence and / or comparison. In some embodiments, a suitable reference object may be an agent, sample, sequence, subject, animal, or individual or a population thereof under conditions that those skilled in the art would consider comparable, for example, for the purpose of assessing one or more specific variables (e.g., the presence or absence of an agent or condition) or a representative measure or characteristic thereof.

[0125] Small molecules:The term "small molecule" is a term used in the art and includes molecules with a molecular weight of less than about 1000 or less than about 500. In one embodiment, a small molecule contains more than just peptide bonds. In another embodiment, a small molecule is not an oligomer. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, peptide mimics, nucleic acids, carbohydrates, and small organic molecules (e.g., polyketide compounds) (Cane et al. (1998)). Science 282:63), and a library of natural product extracts. In another embodiment, the compound is a small organic non-peptide compound. Unless otherwise stated, the term is intended to cover all stereoisomers, geometric isomers, tautomers, and isotopes of the target chemical structure.

[0126] Therapeutic agents: As used herein, the term "therapeutic agent" means any agent that, when administered to a subject, elicits the desired biological and / or pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect in an appropriate population. In some embodiments, an appropriate population may be a group of model organisms (e.g., animal models of a target disease, such as cancer, humanized animal models, animal models containing a human immune system, etc.) or a population. In some embodiments, an appropriate population may be defined by various criteria, such as an age group, sex, genetic background, pre-existing clinical condition, etc. In some embodiments, a therapeutic agent is a substance that can be used to treat a disease, condition, or symptom. In some embodiments, a therapeutic agent is an agent that has been or requires approval from 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.

[0127] Subjects: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, rat, or mouse). In certain embodiments, the subject is a human. In some embodiments, the subject has a disease, condition, or symptom. In some embodiments, the subject is susceptible to a disease, condition, or symptom. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, condition, or symptom. In some embodiments, the subject does not have a disease, condition, or symptom. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, condition, or symptom. In some embodiments, the subject has one or more characteristics that characterize susceptibility to or risk of developing a disease, condition, or symptom. In some embodiments, the subject is a subject who has been tested for and / or treated with a disease, condition, or symptom. In some cases, human subjects may be referred to interchangeably as "patients" or "individuals." Subjects who are given medication related to the treatment of a disease, condition, or symptom in relation to the subject may be referred to as subjects requiring the medication, i.e., subjects in need.

[0128] Effective therapeutic dose: As used herein, "therapeutic effective amount" refers to an amount that produces the desired effect of administration. In some embodiments, the term refers to an amount sufficient to treat a disease, condition, and / or symptom when administered to a population suffering from or susceptible to such a disease, condition, and / or symptom, according to a therapeutic dosing regimen. In some embodiments, a therapeutic effective amount is an amount that reduces the incidence and / or severity and / or delays the onset of one or more symptoms of a disease, condition, and / or symptom. Those skilled in the art will understand that a therapeutic effective amount does not necessarily achieve successful treatment in every particular individual receiving treatment. Rather, a therapeutic effective amount may be an amount that provides a specific, desired pharmacological response in a significant number of subjects when administered to a patient requiring such treatment. In some embodiments, reference to a therapeutic effective amount may refer to an amount measured, such as in one or more specific tissues (e.g., tissues affected by a disease, condition, or symptom) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will understand that in some embodiments, a particular agent or therapy of a therapeutic effective amount may be formulated and / or administered in a single dose. In some implementations, the therapeutic agent may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0129] treat As used herein, the term "treatment" (and "treat" or "treating") means the application of a therapy that partially or completely relieves, improves, reduces, inhibits, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, condition, and / or symptom, or is used to achieve any such outcome. In some embodiments, such treatment may be directed to subjects who do not exhibit signs of the relevant disease, condition, or symptom and / or who exhibit only early signs of the disease, condition, or symptom. Alternatively or additionally, such treatment may be directed to subjects who exhibit one or more definitive signs of the relevant disease, condition, and / or symptom. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, condition, and / or symptom. In some embodiments, treatment may be directed to subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, condition, or symptom.

[0130] II. Multispecific antigen-binding constructs This disclosure includes a variety of binders comprising the domains provided herein, and particularly binders comprising at least one APP-binding domain and at least one masking domain, the masking domain modulating APP binding via the APP-binding domain, wherein the masking domain associates with a protein-hydrolyzable cleavable linker. In some embodiments, the binders covered by this disclosure may comprise at least one target-cell binding domain. In some embodiments, the binders covered by this disclosure may comprise two target-cell binding domains, for example, wherein the two target-cell binding domains target the same epitope and / or antigen, or wherein the two target-cell binding domains target different epitopes and / or antigens.

[0131] In some embodiments, any of the multispecific antigen-binding constructs may be a therapeutic binder. A multispecific antigen-binding construct includes multiple antigen-binding domains for binding antigens for therapeutic purposes. In the embodiments described herein, the multispecific construct includes: a first antigen-binding domain that binds to inhibit antiphagocytic protein (APP); and 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.

[0132] In some embodiments, the provided multispecific antigen-binding construct comprises: (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 comprising a protein-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 comprising the protein-cleavable linker connects 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 connected via a linker comprising the protein-cleavable linker. In some embodiments, the immunoglobulin Fc region and the second antigen-binding domain are connected via a linker comprising the protein-cleavable linker.

[0133] In some embodiments, the binders covered by this disclosure include antibody structures linked to an APP binding domain and a masking 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 four-chain immunoglobulin structure comprising or substantially comprising two immunoglobulin heavy chains and two immunoglobulin light chains, or variants thereof recognized in the art, such as those disclosed herein. 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 by 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 association 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 association of a heavy chain variable domain present in the immunoglobulin heavy chain and a light chain variable domain present in the immunoglobulin light chain. In some embodiments, the Fc domain may be present in an antibody structure comprising two antibody-binding domains, one or each of which is formed by association of heavy and light chain variable domains present in an antigen-binding fragment (Fab) domain. In some embodiments, the Fc domain may be present in an antibody structure comprising two antibody-binding domains, one or each of which is formed by association of heavy and light chain variable domains present in an antibody fragment (such as scFv). In some embodiments, the Fc domain may be present in an antibody structure comprising two antibody-binding domains, one or each of which is present in a variable domain of a heavy-chain-only antibody (VHH).

[0134] In some embodiments, the binder covered by this disclosure includes an antibody structure optionally linked to an APP-binding domain at a first amino acid via a linker, and to a masking domain at a second amino acid via a proteolytically cleavable linker. In some embodiments, the binder covered by this disclosure includes an antibody structure optionally linked to an APP-binding domain at a first amino acid via a linker, the APP-binding domain also being linked to a masking domain at its amino acid (e.g., the terminal amino acid of the APP-binding domain) via a proteolytically cleavable linker. As will be understood from this disclosure, the APP-binding domain and the masking domain are arranged in the binder such that the masking domain can interact (e.g., bind) with the APP-binding domain in a manner that reduces its APP-binding activity.

[0135] In some embodiments, when the adapter containing the protein-hydrolyzable cleavable adapter 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 adapter containing the protein-hydrolyzable cleavable adapter has been protein-hydrolyzed, the second antigen-binding domain does not interfere with the binding of the first antigen-binding domain to APP.

[0136] In some embodiments, the APP-binding domain associates with the antibody structure of the conjugate disclosed herein via an amino acid (e.g., a terminal amino acid) of the Fc domain and / or heavy chain constant domain. In some embodiments, the APP-binding domain associates with the antibody structure of the conjugate disclosed herein via an amino acid (e.g., a terminal amino acid) of the light chain constant domain. In some embodiments, the APP-binding domain associates with the antibody structure of the conjugate disclosed herein via an amino acid (e.g., a terminal amino acid) of the light chain variable domain (e.g., a light chain variable domain present in the Fab). In some embodiments, the APP-binding domain associates with the antibody structure of the conjugate disclosed herein via a protein-hydrolyzable cleavable linker.

[0137] In some embodiments, the target cell binding domain associates with the antibody structure of the conjugate disclosed herein via an amino acid (e.g., a terminal amino acid) of the Fc domain and / or heavy chain constant domain. In some embodiments, the target cell binding domain associates with the antibody structure of the conjugate disclosed herein via an amino acid (e.g., a terminal amino acid) of the light chain constant domain. In some embodiments, the target cell binding domain associates with the antibody structure of the conjugate disclosed herein via an amino acid (e.g., a terminal amino acid) of the light chain variable domain (e.g., a light chain variable domain present in Fab).

[0138] In some embodiments, the target cell binding domain associates with the antibody structure of the conjugate disclosed herein via a linker that does not contain motifs known to be cleaved and / or substantially cleaved by human proteases.

[0139] In some embodiments, the target cell binding domain associates with the antibody structure of the conjugate disclosed herein via a linker that does not contain motifs known to be cleaved and / or substantially cleaved by human proteases in the therapeutic target microenvironment.

[0140] In some embodiments, the target cell binding domain associates with the antibody structure of the conjugate disclosed herein via a linker that does not contain motifs known to be cleaved and / or substantially cleaved by proteases, which cleave and / or substantially cleave the proteolytically cleavable linker associated with the APP binding domain. In some embodiments, the target cell binding domain associates with the antibody structure of the conjugate disclosed herein via a linker that does not contain motifs known to be cleaved and / or substantially cleaved by proteases, which cleave and / or substantially cleave the proteolytically cleavable linker associated with the APP binding domain.

[0141] In some embodiments, the binders covered by this disclosure are labeled for detection. In some embodiments, construct labeling encompasses direct labeling of the construct by coupling (i.e., physical linking) to a detectable substance, and indirect labeling of antibodies by reactivity with the detectable substance. Labeling and methods used for labeling are well known in the art and include, but are not limited to: radioactive agents, radioisotopes, fluorescent compounds, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), or indocyanine (Cy5)), chemiluminescent compounds, enzymes, enzyme cofactors, or any other labeling 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); anthocyanins and their derivatives (e.g., indolecarbonyl cyanine, oxacarbonyl cyanine, thiocarbonyl cyanine, and thiocyanine); rhodamine; Oregon green; eosin; Texas red; Nile red; Nile blue; cresol violet; oxazine 170; proflavin; acridine orange; acridine yellow; auramine; crystal violet; malachite green; porphyrin; phthalocyanine; bilirubin; allophycocyanin (APC); green fluorescent protein (GFP) and its variants (e.g., yellow fluorescent protein YFP, blue fluorescent protein BFP, and cyan fluorescent protein CFP); ALEXIFLOUR® compounds (Thermo Fisher Scientific, Waltham, MA); and quantum dots. Other conjugates that can be used as labels include biotin, avidin, and streptavidin.

[0142] A. APP combined with structural domain The binders covered by this disclosure include a domain that binds to an antiphagocytic protein (APP) (the APP-binding domain). Phagocytosis is a natural process used to remove dying or pathogenic cells. Several mechanisms limit the frequency of phagocytosis of healthy cells, one of which is the balance between surface-expressed signaling molecules that promote or activate phagocytosis (sometimes called "eat me" signals) and other signaling molecules that inhibit phagocytosis (sometimes called "don't eat me" signals). However, when the "don't eat me" signals that inhibit phagocytosis are expressed by diseased cells, they can cause, contribute to, and / or exacerbate disease by allowing diseased cells to evade phagocytosis.

[0143] The "eat me" signaling pathway includes antibodies and complement opsonins, exposed phosphatidylserine (PS), calreticulin, oxidized low-density lipoprotein, cell-bound platelet-reactive protein (TSP), modified intracellular adhesion molecule ICAM-3, annexin I, and other modifications to surface proteins. The "don't eat me" signaling pathway may include antiphagocytic receptors mediating the recognition of the "don't eat me" signal. For example, the CD47-SIRPα axis is one of the most studied "don't eat me" checkpoints. In various cancers, cancer cells can overexpress CD47 and / or CD24, enabling them to immunely evade macrophages.

[0144] Proteins involved in the "Don't Eat Me" signaling pathway may be referred to as APPs. In some embodiments, APPs are expressed on myeloid cells. In some embodiments, APPs are expressed on cells targeted for phagocytosis (e.g., cancer cells). In some embodiments, APPs are proteins other than receptors or ligands in the "Don't Eat Me" signaling pathway that inhibit the phagocytic function of myeloid cells (e.g., LILRB2), and such APPs may be similarly expressed on myeloid cells or on cells targeted for phagocytosis (e.g., cancer cells).

[0145] Unwilling to be bound by any particular scientific theory, CD47 expression on cells acts as a marker of its own, inhibiting phagocytosis through its interaction with SIRPα. CD47 (also known as integrin-associated protein, OV-3, and Rh-associated protein) is a conserved, universally expressed 45–55 kDa transmembrane glycoprotein belonging to the Ig superfamily. CD47 possesses a single N-terminal extracellular immunoglobulin variable region (IgV) domain, followed by five hydrophobic transmembrane segments and a short C-terminal cytoplasmic tail, which are alternately spliced ​​to form four isotypes. 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 and is enriched in, for example, monocytes, macrophages, CD8α classical type II dendritic cells (cDC2), neutrophils and osteoclasts, as well as microglia and neurons.

[0146] Besides SIRPα and CD47, other receptors and ligands can also inhibit the function of immune cells, such as myeloid cells. For example, CD31 (also known as PECAM-1) and inhibitory receptors of the CD300 family, including CD300a and CD300f, can inhibit phagocytosis. CD300a and CD300f are expressed on myeloid cells and bind ligands such as phosphatidylserine (PS).

[0147] Siglec, or sialic acid-binding Ig-like lectins, is a large family of receptors that can inhibit phagocytosis and includes, for example, CD33 (also known as Siglec-3), CD22 (also known as Siglec-2), and SIGLEC10. CD33 is expressed, for example, on myeloid cells and microglia. CD22 is expressed, for example, on B cells, as well as myeloid-derived cells and microglia. SIGLEC10 is expressed on myeloid cells and some lymphocytes and participates in signal transduction axes along with CD24.

[0148] PD-1 (also known as programmed cell death protein 1 and CD279) is a well-known immunosuppressive receptor with two homologous ligands, called 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 expression on lymphocytes, PD-1 expression can also be induced in macrophages, for example, through infection.

[0149] Human LILRB1 (leukocyte immunoglobulin-like receptor B1; also known as CD85J, ILT2, LIR-1) is an inhibitory receptor expressed on myeloid cells 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 being bound by any particular scientific theory, the β2-microglobulin (B2M) subunit of the MHC class I complex mediates the interaction between MHC class I and LILRB1. MHC class I expression can protect cells from phagocytosis by binding to LILRB1.

[0150] In some implementations, the APP covered by this disclosure is combined with the domains listed in the following table: Table 1

[0151] *Table 1 includes: RNA nucleic acid molecules (e.g., thymine replaced by uridine); nucleic acid molecules encoding orthologs of the encoded protein; DNA or RNA nucleic acid sequences comprising nucleic acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher identity in their full length with any of the nucleic acid sequences or portions listed in Table 1; orthologs of any of the proteins listed in Table 1; and amino acid sequences comprising amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher identity in their full length with any of the amino acid sequences or portions listed in Table 1. These nucleic acid or amino acid moieties can function as full-length nucleic acid or amino acid molecules, as further described herein.

[0152] In some embodiments, the APP binding domains covered 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 pair (e.g., binding pair expressed by or present on myeloid cells, such as the binding pair disclosed herein).

[0153] In some embodiments, the APP binding domains covered 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 pair (e.g., binding pairs expressed or present thereon by cells (such as cancer cells) targeted to regulate (e.g., direct and / or indirect killing), such as the binding pairs disclosed herein).

[0154] In some embodiments, the APP-binding domain covered 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 killing) and SIRPα expressed on myeloid cells (e.g., macrophages, dendritic cells, monocytes, or neutrophils). In some embodiments, the APP-binding domain covered 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 killing) and SIGLEC10 expressed on myeloid cells (e.g., macrophages, dendritic cells, monocytes, or neutrophils). In some embodiments, the APP-binding domain covered by this disclosure binds to PD-1 ligands, such as PD-L1 and / or PD-L2 or PD-1. In some embodiments, the APP-binding domain inhibits the interaction between PD-1 ligands expressed on cells targeted for regulation (e.g., direct and / or indirect killing) and PD-1 expressed on myeloid cells (e.g., macrophages, dendritic cells, monocytes, or neutrophils). In some embodiments, the APP-binding domain covered by this disclosure binds to β2-microglobulin (B2M) or MHC-I or LILRB1. In some embodiments, the APP-binding domain inhibits the interaction between B2M or MHC-I expressed on cells targeted for regulation (e.g., direct and / or indirect killing) 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 APP expressed on myeloid cells. In some implementations, 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 suppressor cells (MDSCs)) to block the inhibition of phagocytosis by myeloid cells.

[0155] 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) an extracellular domain (ECD) of a 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, the variant being engineered to improve binding to APP. In some embodiments, the binding fragment of the cell surface-expressed protein includes a portion of the ECD of the cell surface-expressed protein. In some embodiments, the binding fragment of the cell surface-expressed protein includes the immunoglobulin variable (V) region (domain 1) of the ECD of the cell surface-expressed protein. In some embodiments, the binding fragment of the cell surface-expressed protein consists substantially of the immunoglobulin variable (V) region (domain 1) of the ECD of the cell surface-expressed protein.

[0156] In some embodiments, the multispecific antigen-binding construct comprises: a first antigen-binding domain comprising: (i) a domain that binds to wild-type SIRPα of an antiphagocytic protein (APP), or (ii) a variant thereof comprising one or more amino acid substitutions in the wild-type SIRPα domain that improve binding to APP, wherein APP is CD47; and a second antigen-binding domain, which is an anti-SIRPα antibody or an 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 antigen-binding domain and the second antigen-binding domain are linked by a proteolytically cleavable linker.

[0157] In some embodiments, the APP-binding domain binds to APP expressed and / or associated with the therapeutic target cells. Binders containing the APP-binding domain, as covered by this disclosure, can induce phagocytosis by the target cells.

[0158] In some embodiments, the binders covered by this disclosure include an APP-binding domain that binds to an APP expressed by a cell or cell type that is associated with, characterized by, represents, causes, contributes to, and / or phagocytoses the target symptom in a manner conducive to the treatment of the symptom. In some embodiments, the target symptom is cancer. In some embodiments, the cancer is selected from, but is not limited to: adenocarcinoma, bile duct (cholangiocarcinoma), bladder cancer, bone cancer, breast cancer (e.g., triple-negative breast cancer or Her2-negative breast cancer), carcinoid, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, glioma, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), 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 implementations, the cancer is selected from, but is 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); head and / or neck cancer, breast cancer, cancers of the circulatory system (e.g., heart, mediastinum, and pleura, as well as other intrathoracic organs, vascular cancer, and tumor-associated vascular tissue); cancers of the blood and lymphatic system (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt lymphoma, AIDS-related lymphoma, malignant immunoproliferative disorders, multiple myeloma and malignant plasma cell tumors, lymphoid leukemia). Blood disorders, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other specific cell types of leukemia, leukemia of unknown cell type, unspecified malignant tumors 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 organs); cancers of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectum, rectum, rectosigmoid junction, rectum, anus and anal canal); cancers involving the liver and intrahepatic bile ducts, gallbladder and gallbladder. Cancers of other parts of the digestive tract, pancreas, and other digestive organs; cancers of the oral cavity (e.g., lips, tongue, gums, floor of mouth, palate, parotid glands, salivary glands, tonsils, oropharynx, nasopharynx, pyriform recess, 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 female reproductive organs, placenta, penis, prostate, testes, and other parts associated with male reproductive organs); cancers of the respiratory tract (e.g., nasal cavity, middle ear, paranasal 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., skeletal system ... For example, cancers of the bones and articular cartilage of the limbs, joint 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 involving other tissues (including the peripheral and autonomic nervous systems, connective and soft tissues, retroperitoneal space and peritoneum, eyes and appendages, thyroid gland, adrenal glands and other endocrine glands and related structures), secondary and unspecified malignancies of the lymph nodes, secondary malignancies of the respiratory and digestive systems, and secondary malignancies of other sites. In some embodiments, the condition is selected from, for example, infections (e.g., bacterial and / or viral infections), atherosclerosis, cardiovascular diseases (e.g., heart failure after myocardial infarction), autoimmune diseases (e.g., systemic lupus erythematosus (SLE), autoimmune nephritis, autoimmune uveitis, or autoimmune valvular heart disease), organ transplant rejection, fibrotic diseases, or neurological diseases. In some embodiments, the binders covered by this disclosure include an APP-binding domain that binds to APP expressed by additional cells or cell types characterized by expressing target cell antigens covered by this disclosure.

[0159] 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 an 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.

[0160] In some embodiments, the APP-binding domain is a 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 a 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 a 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 a 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 a 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 a 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 a 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).

[0161] In some embodiments, the APP-binding domain may comprise an antibody or its antigen-binding portion. In some embodiments, the APP-binding antibody or its antigen-binding portion may comprise 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 portion may comprise 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 portion may comprise 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-binding antibody or its antigen-binding portion may be or include: an extracellular domain (ECD) of a cell surface-expressed protein, a mutant form (variant) of an ECD of a cell surface-expressed protein engineered to improve binding to a target, an intracellular antibody, a domain antibody, an antibody mimic, Zybody®, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd' fragment, an Fd fragment, an isolated CDR or a group thereof, a single-chain antibody, a single-chain Fv (scFv), or a disulfide-linked Fv. (sdFv), peptide-Fc fusions, single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof), camel antibodies, camel-derived antibodies, masking antibodies (e.g., Probody®), affybody, anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), single-chain or tandem biantibodies (TandAb®), VHH, Anticalin®, Nanobody® small antibodies, BiTE®, ankyrin repeat protein 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 foregoing.

[0162] In some embodiments, the APP-binding domain is a variant APP-binding domain comprising one or more amino acid substitutions that improve binding to CD47. In some embodiments, the binding of the variant APP-binding domain comprising one or more amino acid substitutions in the IgV domain to CD47 is improved compared to binding to the wild-type APP-IgV domain. In some embodiments, the APP-binding domain has a dissociation constant (K0) of less than 100 picomolars (pM). D ) binds to CD47. In some instances, the variant APP binds to the domain with a dissociation constant (K0) of less than 100 picomoles (pM).D Combined with CD47.

[0163] In some implementations, the APP covered by this disclosure combines the affinity (K) of the structural domain to the target APP. D The affinity can be from about 0.002 to about 200 nM. In some embodiments, the binding affinity of the APP binding domain covered by this disclosure to the target APP can be any of the following values: 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 lower. In some embodiments, the binding affinity is below any of the following values: about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5 pM, or about 2 pM or lower, or any range therebetween, such as about 5 nM to about 35 nM. In some embodiments, the binding affinity of the APP binding domain covered by this disclosure to the target APP may be about less than 1 × 10⁻⁶. -7 M, such as approximately less than 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or lower. In some embodiments, affinity (K) can be determined using methods well known in the art. D ), such as by using surface plasmon resonance (SPR) technology in BIACORE® measuring instruments.

[0164] 1. CD47 APP combined with structural domain Blocking the interaction between CD47 and endogenous SIRPα expressed on the surface of macrophages and dendritic cells can prevent CD47 / SIRPα-mediated signaling, thereby eliminating CD47 / SIRPα-mediated inhibition of phagocytosis. Existing regulators of this pathway typically target the ubiquitously expressed CD47 cell surface molecule (see, for example, 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, the CD47 APP-binding domain can be used to inhibit or block the interaction of CD47 with endogenous or wild-type SIRPα. For example, the CD47 APP-binding domain binding to CD47 enables phagocytosis of solid tumor cells, inhibits tumor growth, and prevents tumor cell metastasis. This article provides a multispecific antigen-binding construct containing the CD47 APP-binding domain. These multispecific constructs are specific for CD47 via a CD47-binding domain and may also include a binding domain specific for another antigen. In some embodiments, the multispecific antigen-binding construct contains a CD47 APP-binding domain comprising a SIRPα peptide 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 peptide. In these embodiments, the multispecific antigen-binding construct may contain a CD47 APP-binding domain as 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.

[0165] (i) SIRPα SIRPα protein is a membrane glycoprotein expressed by neurons and myeloid cells, and is enriched 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 interacts 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 under Uniprot accession number P78324.

[0166] CD47 functions as a ligand for SIRPα. Upon binding of CD47 to endogenous SIRPα on the surface of macrophages, SIRPα initiates signaling to inhibit phagocytosis by CD47-containing cells. The binding interface between SIRPα and CD47, and the residues of the two proteins involved in the binding, are known and have been previously 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 multispecific antigen-binding constructs comprising SIRPα peptides, such as variant SIRPα peptides (e.g., variant SIRPα peptides with enhanced 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 peptides (such as the SIRPα peptides presented herein) can bind CD47 and inhibit the interaction of CD47 with endogenous or wild-type SIRPα.

[0167] In some embodiments, the multispecific antigen-binding construct comprises an antigen-binding protein that binds to an anti-phagocytic protein (APP) as CD47, such as an antigen-binding protein as SIRPα. In the embodiments described herein, the antigen-binding protein is SIRPα, which binds to an anti-phagocytic protein (APP) as CD47. In some embodiments, the SIRPα peptide 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 a higher affinity than CD47 on non-lesioned cells.

[0168] In some embodiments, the SIRPα in the multispecific antigen-binding construct provided herein inhibits the binding of the extracellular domain of the wild-type SIRPα peptide or its binding fragment to CD47. In some embodiments, the SIRPα provided herein inhibits the binding of wild-type SIRPα to the IgSF domain of the CD47 protein. In the embodiments described herein, CD47 is the human CD47 protein.

[0169] In the implementation scheme, the SIRPα in the multispecific antigen binding construct comprises a SIRPα polypeptide selected from: (i) the ECD of wild-type SIRPα; (ii) the binding fragment of wild-type SIRPα; and (iii) a variant of the ECD or binding fragment of wild-type SIRPα, which is engineered to improve binding to CD47.

[0170] In some embodiments, the multispecific antigen-binding construct comprises a wild-type SIRPα polypeptide or a portion thereof. In some embodiments, the SIRPα in the multispecific antigen-binding construct comprises a domain of wild-type SIRPα. In some embodiments, the SIRPα in the multispecific antigen-binding construct comprises the extracellular domain (ECD) of wild-type SIRPα. In some embodiments, the SIRPα polypeptide is a wild-type SIRPα polypeptide composed essentially of 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 (IgV; also known as D1) of the ECD of SIRPα. In some embodiments, the SIRPα polypeptide is a binding fragment of a wild-type SIRPα polypeptide that comprises the SIRPα IgV domain and binds to CD47. In any of the foregoing embodiments, the SIRPα polypeptide is an APP-binding domain.

[0171] In some embodiments, the multispecific antigen-binding construct comprises a variant SIRPα polypeptide. The variant SIRPα polypeptide may contain one or more amino acid modifications in an unmodified SIRPα polypeptide (such as a wild-type SIRPα polypeptide, such as any wild-type SIRPα provided herein). For example, the variant SIRPα polypeptide provided herein contains one or more amino acid substitutions (alternatively, “mutations” or “replacements”), deletions, or additions in an unmodified SIRPα polypeptide (such as a wild-type SIRPα polypeptide 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 (extracellular domain) of a reference (e.g., unmodified or wild-type) SIRPα sequence. In some embodiments, one or more amino acid modifications are located in D1 of SIRPα.

[0172] Unless otherwise stated, as indicated throughout this disclosure, amino acid modifications in the variant SIRPα polypeptide are designated by amino acid position numbers corresponding to the position numbers of the unmodified or wild-type SIRPα ECD sequence shown in SEQ ID NO: 206 or a portion thereof shown in SEQ ID NO: 103 or 104. Identifying the corresponding position of a modification (e.g., amino acid substitution) in the SIRPα polypeptide (including the portion containing its IgV domain) is within the capabilities of those skilled in the art, such as by comparing the variant SIRPα sequence with the amino acid sequence shown in SEQ ID NO: 103 or 104. Throughout this disclosure, when referring to amino acid substitutions, the amino acid position is indicated in the middle, with the corresponding reference (e.g., unmodified or wild-type) amino acid listed before the number, and the variant amino acid substitution listed after the number. For example, amino acid substitution N80A means replacing asparagine (N) with alanine (A) at position 80, where position 80 is the 80th amino acid in the amino acid sequence shown in SEQ ID NO: 103 or 104.

[0173] In some embodiments, the SIRPα with introduced amino acid modifications is a wild-type SIRPα containing the extracellular domain of SIRPα, such as any wild-type SIRPα described herein. In exemplary embodiments, the variant SIRPα contains one or more amino acid modifications in a wild-type SIRPα containing the extracellular domain of SIRPα. However, the variant SIRPα polypeptide does not need to contain the entire extracellular domain (ECD). In some embodiments, the variant SIRPα contains one or more amino acid modifications in a 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 located in the SIRPα ECD or its IgV-containing portion. In some embodiments, the variant SIRPα contains one or more amino acid modifications in a wild-type SIRPα containing the IgV domain of SIRPα. In some embodiments, the variant SIRPα contains one or more amino acid modifications of a binding fragment of the SIRPα ECD containing D1. In some embodiments, the variant SIRPα contains one or more amino acid modifications in the amino acid sequence shown in SEQ ID NO. 103 or 104. In some embodiments, the variant SIRPα includes one or more amino acid modifications in the amino acid sequence shown in SEQ ID NO. 103.

[0174] In some implementations, the variant SIRPα peptide is a soluble peptide and lacks a transmembrane domain.

[0175] In embodiments described herein, the variant SIRPα peptide exhibits altered (e.g., enhanced) binding affinity for CD47. In some embodiments, the variant SIRPα binds CD47 with a higher affinity than wild-type SIRPα. In embodiments described herein, 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 improve binding to CD47. In a particular embodiment, the variant SIRPα includes one or more amino acid substitutions in the extracellular domain of wild-type SIRPα or the binding fragment of SIRPαECD, which improves binding to CD47 compared to binding of wild-type SIRPα without such one or more amino acid substitutions. In embodiments described herein, the variant SIRPα includes a binding fragment of wild-type SIRPα engineered to improve binding to CD47. In embodiments, the binding fragment of SIRPα includes D1. In the embodiments described herein, the variant SIRPα comprises an extracellular domain of wild-type SIRPα engineered to improve binding to CD47. In the embodiments described herein, the variant SIRPα essentially consists of an extracellular domain of wild-type SIRPα engineered to improve binding to CD47. The improved binding of the variant SIRPα compared to wild-type or unmodified SIRPα is demonstrated by the variant SIRPα exhibiting a higher affinity for CD47 compared to wild-type SIRPα. The higher affinity of the variant SIRPα compared to wild-type SIRPα is attributable to a smaller dissociation constant (K). D This can be demonstrated by [the following]. Therefore, the smaller the dissociation constant, the tighter the binding of 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, the variant SIRPα binds to CD47 on diseased cells (e.g., tumor cells) with a higher affinity than CD47 on non-pathological cells.

[0176] In some embodiments, the SIRPα (such as variant SIRPα) provided herein binds to CD47, wherein the binding of wild-type or endogenous SIRPα to CD47 is blocked. For example, variant SIRPα and wild-type SIRPα (e.g., endogenous) peptides may “compete” for the same CD47 epitope. In some instances, variant SIRPα or wild-type SIRPα in a multispecific antigen-binding construct may outperform wild-type or endogenous SIRPα in binding to CD47. In some embodiments where variant SIRPα outperforms wild-type (e.g., endogenous) SIRPα in binding to CD47, the variant SIRPα exhibits enhanced affinity for CD47 compared to wild-type (e.g., endogenous) SIRPα.

[0177] In embodiments where the SIRPα polypeptide includes a SIRPα binding fragment, the fragment or portion of the SIRPα polypeptide is sufficient to bind CD47. In some embodiments, the specific binding fragment is shorter than the full-length ECD shown in SEQ ID NO: 206. In some embodiments, the SIRPα binding fragment (also referred to herein as the SIRPα binding region) includes the immunoglobulin variable (V) region (domain 1) of the SIRPα ECD. 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 consists essentially of the immunoglobulin variable (V) region (domain 1) of the SIRPα ECD. In some embodiments, the SIRPα binding fragment consists essentially of the sequence shown in SEQ ID NO. 103. In some embodiments, the SIRPα binding fragment contains / consistently consists of 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 1-115 of SEQ ID NO: 206.

[0178] In some embodiments, SIRPα is a SIRPα binding region of 100 to 120 amino acids in length, such as 105 to 115, 105 to 110, 105 to 115, 110 to 120, 110 to 115, or 115 to 120 amino acids in length. In some embodiments, the SIRPα binding region is 106 to 118 amino acids in length. In some embodiments, the SIRPα binding region is 112 to 118 amino acids in length. In the embodiments described herein, the SIRPα binding region is or is about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 amino acids in length. In some instances, the SIRPα binding region is or is about 115 amino acids in length.

[0179] In some embodiments, wild-type SIRPα is wild-type human SIRPα. In some embodiments, variant SIRPα contains one or more amino acid modifications, such as one or more amino acid substitutions like those in wild-type human SIRPα. In the 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 the embodiments herein, the IgV domain of wild-type human SIRPα is shown in SEQ ID NO. 103 and SEQ ID NO. 104. In the embodiments herein, the ECD of wild-type human SIRPα is shown in SEQ ID NO. 206.

[0180] In some embodiments, the variant SIRPα polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, such as substitutions, in the wild-type SIRPα sequence. In some embodiments, the variant SIRPα polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions in the ECD of the wild-type TACI sequence or its specific binding fragment. The modifications (e.g., substitutions) may be located in the IgV domain. In some embodiments, the variant SIRPα polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions in the IgV domain of the wild-type SIRPα sequence.

[0181] 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. 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 the SIRPα ECD containing the IgV domain of SIRPα. In a particular embodiment, 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 an 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.

[0182] 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. In some embodiments, SIRPα 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.

[0183] 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. In some embodiments, SIRPα 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.

[0184] In some embodiments, variants of SIRPα containing one or more amino acid modifications (e.g., amino acid substitutions) as described have 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. [Domains]

[0185] In some embodiments, variant SIRPα containing one or more amino acid modifications (e.g., amino acid substitutions) as described have 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.

[0186] In some embodiments, SIRPα is a variant of SIRPα that includes one or more amino acid substitutions in the IgV domain of wild-type SIRPα to improve binding to CD47. In some embodiments, the binding of the variant SIRPα containing one or more amino acid substitutions in the IgV domain to CD47 is improved compared to the binding of wild-type SIRPα to the IgV domain.

[0187] In some implementations, the SIRPα binding domains have a certain dissociation constant (K). DSIRPα 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 masking domain to be used. In some implementations, the binding affinity of the SIRPα binding domain (such as the variant SIRPα binding domain) to CD47 is any of the following values: 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 lower. In some embodiments, the binding affinity is below any of the following values: about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5 pM, or about 2 pM or lower, or any range therebetween, such as about 5 nM to about 35 nM. In some embodiments, the binding affinity of variant SIRPα to CD47 is or is about less than 1 × 10⁻⁶. -7 M, such as approximately less than 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or lower. In some embodiments, affinity (K) can be determined using methods well known in the art. D ), such as by using surface plasmon resonance (SPR) technology in BIACORE® measuring instruments.

[0188] For example, in some embodiments, the SIRPα binding domain is a wild-type SIRPα binding domain or a variant thereof, wherein the dissociation constant (K) for binding to wild-type human CD47 (such as CD47 expressed on the cell surface) is... D (Below 100 nanomolars (nM).) In some implementations, K DThe ranges are 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.

[0189] In some embodiments, the SIRPα binding domain is a variant SIRPα that includes one or more amino acid substitutions in the wild-type SIRPα domain (e.g., the IgV domain) to improve binding to CD47. In some embodiments, the variant SIRPα has a moderate affinity for binding to CD47. In some embodiments, the variant SIRPα has a dissociation constant (K0) of less than 1 nM. D ) binds to wild-type human CD47, such as SIRPα expressed on the cell surface. In some implementations, K D The values ​​are 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. An example of this variant SIRPα is SIRPα containing the E54Q mutation, which in some cases is the only mutation in the domain relative to wild-type SIRPα.

[0190] In some embodiments, the SIRPα binding domain is a variant SIRPα that includes one or more amino acid substitutions in the wild-type SIRPα domain (e.g., the IgV domain) to improve binding to CD47. In some embodiments, the variant SIRPα has a high or relatively high affinity for binding to CD47. In some embodiments, the variant SIRPα has a dissociation constant (Ki) of less than 100 picomolar (pM). D ) binds to wild-type human CD47, such as SIRPα expressed on the cell surface. In some implementations, K DThe range is 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. An example of this variant is the variant SIRPα, known as CV1. In some embodiments, the variant SIRPα has amino acid substitutions V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), and V92I, corresponding to amino acid numbers in SEQ ID NO: 103 or SEQ ID NO: 104.

[0191] In some implementations, the SIRPα binding domain (such as the variant SIRPα binding domain) binds CD47, whose dissociation constant (K) D The value is approximately 1 pM to 100 pM, or optionally approximately 10 pM to 50 pM.

[0192] The variant SIRPα peptides provided herein may comprise one or more amino acid modifications known in the art to alter (e.g., improve) the binding of the peptide to CD47. Exemplary amino acid modifications (e.g., substitutions) are described in Lee et al., J Immunol. 1 Dec 2007; 179(11):7741-50; Weiskopf et al., Science. 5 July 2013; 341(6141):10.1126 / science.1238856; International Patent Publication No. WO 2016 / 023040). In some embodiments, the variant SIRPα comprises amino acid substitutions in the residues in contact with CD47. In some embodiments, variant SIRPα contains an amino acid substitution at one or more of the following positions in the contact residues: 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 contains an amino acid substitution in the hydrophobic core. In some embodiments, variant SIRPα contains an amino acid substitution at one or more of the following positions in the hydrophobic core: L4, V6, V27, I36, F39, L48, I49, Y50, F57, V60, M72, F74, I76, V92, F94, and F103.In some embodiments, variant SIRPα comprises a combination of amino acid substitutions selected from: 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, S66T 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, A21 V, V27I or V27L, I31T, I31S or I31F, E47V or E47L, K53R, E54Q, H56P or H56R, S66T or S66G, F94L or F94V and F103V; L4V or L4I, V6I or V6L, V27I or V27L, I31T, I31 S 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; V6I, V27I, I31F, E47L, K53R, E54Q, H56P and S66T; L4V, V6I, V27I, 131 F, 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.

[0193] In some embodiments, the variant SIRPα polypeptide contains one or more amino acid substitutions selected from the wild-type SIRPα polypeptide or its specific binding fragment: 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 conserved amino acid substitutions thereof, corresponding to the amino acid number of SEQ ID NO. 103. In some embodiments, the variant SIRPα polypeptide contains one or more amino acid substitutions selected from the wild-type SIRPα polypeptide or its specific binding fragment: 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 conserved amino acid substitutions thereof, corresponding to the amino acid number of SEQ ID NO. 104.

[0194] Conservative amino acid modifications, such as substitutions, are any amino acid belonging to the same class as the substituted amino acid, rather than a 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).

[0195] In some embodiments, the variant SIRPα peptide has one or more amino acid substitutions in the wild-type SIRPα peptide or the specific binding fragment, wherein the one or more amino acid substitutions are K53R, E54Q, and S66T, corresponding to the amino acid numbers of SEQ ID NO: 103.

[0196] In some embodiments, the variant SIRPα peptide has one or more amino acid substitutions in the wild-type SIRPα peptide or the specific binding fragment, wherein the one or more amino acid substitutions are K53R, E54Q, and L66T, corresponding to the amino acid numbers of SEQ ID NO: 104.

[0197] In some embodiments, the variant SIRPα polypeptide contains one or more amino acid substitutions in the wild-type SIRPα polypeptide or the specific binding, wherein the one or more substitutions are V6I, V27I, I31F, E47V, K53R, E54Q, H56P, S66T, and V92I; or V6I, V27I, I31F, E47L, K53R, E54Q, H56P, 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 numbers of SEQ ID NO: 103.

[0198] In some embodiments, the variant SIRPα polypeptide contains one or more amino acid substitutions in the wild-type SIRPα polypeptide or the specific binding, wherein the one or more substitutions are V6I, A27I, I31F, E47V, K53R, E54Q, H56P, L66T, and V92I; or V6I, A27I, I31F, E47L, K53R, E54Q, H56P, 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 numbers of SEQ ID NO: 104.

[0199] In some embodiments, the variant SIRPα polypeptide contains one or more amino acid substitutions in the wild-type SIRPα polypeptide or the specific binding, wherein the one or more substitutions are V6I, V27I, I31F, E47V, K53R, E54Q, H56P, S66T and V92I, corresponding to the amino acid numbers of SEQ ID NO: 103.

[0200] In some embodiments, the variant SIRPα polypeptide contains one or more amino acid substitutions in the wild-type SIRPα polypeptide or the specific binding, wherein the one or more substitutions are V6I, A27I, I31F, E47V, K53R, E54Q, H56P, L66T and V92I, corresponding to the amino acid numbers of SEQ ID NO: 104.

[0201] 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.

[0202] 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.

[0203] In some embodiments, the variant SIRPα polypeptide comprises the amino acid sequence shown in SEQ ID NO: 105.

[0204] In some embodiments, the variant SIRPα polypeptide is represented by the amino acid sequence shown in SEQ ID NO: 105.

[0205] In some implementations, the SIRPα peptide is deglycosylated.

[0206] In some embodiments, SIRPα contains an N-glycosylation site N80 in the D1 region, which is mutated to alanine (A). In other embodiments, the N-glycosylation site N80 is not mutated, and the glycosylation site is retained.

[0207] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in the wild-type SIRPα polypeptide or the specifically bound polypeptide, wherein the one or more substitutions include N80A, corresponding to the amino acid number of SEQ ID NO: 103 or SEQ ID NO: 104.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] In some embodiments, the variant SIRPα polypeptide comprises the amino acid sequence shown in SEQ ID NO: 27.

[0213] In some embodiments, the variant SIRPα polypeptide is represented by the amino acid sequence shown in SEQ ID NO: 27.

[0214] (ii) Anti-CD47 antibody It has been previously demonstrated that blocking CD47 activity with anti-CD47 antibodies can activate the phagocytosis of CD47+ cells (such as tumor cells) by macrophages. Administration of anti-CD47 monoclonal antibodies can reduce tumor burden in mouse models of hematologic malignancies and solid tumors. In embodiments herein, multispecific antigen-binding constructs may contain an anti-CD47 antibody or an antigen-binding fragment thereof. For example, previously developed and / or known anti-CD47 antibodies may be included in the multispecific antigen-binding constructs provided herein. In some embodiments, the anti-CD47 antibody of this disclosure blocks the binding between the extracellular domain of the SIRP-α peptide (e.g., an ECD containing a D1 domain) and the IgSF domain of the human CD47 peptide. For example, the anti-CD47 antibody and the SIRP-α peptide 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.

[0215] Any of the numerous publicly available and / or known anti-CD47 agents can 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 molorizumab and 5F9, 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, a humanized IgF4 anti-CD47 monoclonal antibody (Celegene, see ClinicalTrials.gov identifiers NCT02641002 and NCT02367196, and Narla. Abstract 4694, Immunology. 2017 4694-4694); AO-176, a humanized IgF2 anti-CD37 mAb (Arch Oncology; see ClinicalTrials.gov identifier 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); SRF231 human IgG4 anti-CD47 mAb (SurfaceOncology, see ClinicalTrials.gov ID NCT03512340; Holland. Blood. 2016;128(22) 1843-1843); IMC-002, fully human IgG4 anti-CD47 mAb (ImmuneOncia Therapeutics; see ClinicalTrials.gov ID NCT04306224; Yoo, J. Immunother. Cancer. 2020;8 (Supplement 3) A237-A237); litelimab, humanized anti-CD47 antibody (Innovent Biologics; see ClinicalTrials.gov (ID NCT0376149); SHR-1603 (Jiangsu Hengrui Medicine, Co., Ltd.); TJC4 (I-Mab Biopharma, Co., Ltd.); IBI188 (Innovent Biologics, Inc.); and AO-176 (Arch Oncology, Inc.); or any variant or combination thereof.In other instances, previously demonstrated anti-CD47 monoclonal antibodies that block CD47-SIRP interaction may be included in the multispecific antigen-binding constructs provided herein, including B6H12.2 and BRIC126 (see, for example, Subramanian et al., Blood. 2006; 107:2548–2556). In other instances, the multispecific antigen-binding constructs provided herein may contain Hu5F9-G4, a humanized IgG4 monoclonal antibody that shows blockade of CD47-SIRPα interaction (ClinicalTrials.gov ID NCT02953509). Anti-CD47 antibodies or antibody fragments. 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 embodiments, previously developed VHH domains may be included in the multispecific antigen-binding constructs provided herein. For example, HuNb1, which is a VHH with high affinity for CD47.

[0216] In the embodiments described herein, the multispecific antigen-binding construct may contain a pan-reactive anti-CD47 antibody or an antigen-binding fragment thereof. For example, the multispecific antigen-binding construct provided herein contains an anti-CD47 antibody or an antigen-binding fragment thereof that has affinity for wild-type CD47 and also for other CD47 variants.

[0217] B. Masking domain The binding agents covered by this disclosure may include a masking domain that modulates the binding of the APP-binding domain to APP. In some embodiments, the masking domain binds to the APP-binding domain in a manner that inhibits the binding of the APP-binding domain to APP upon binding. This disclosure also includes the masking domain being able to associate with a proteolytically cleavable linker, such that when associated with the binding agent (i.e., when the linker is not cleaved), the masking domain inhibits APP binding, but when the linker is cleaved, the inhibition of APP binding is released, thereby achieving activatable activity. Therefore, the binding agents covered by this disclosure, comprising an APP-binding domain and a masking domain that binds to the APP-binding domain, may modulate myeloid cell activity depending on the cleavage of the proteolytically cleavable linker. Furthermore, the activity of the binding agent may be specific to a particular microenvironment when one or more proteases capable of cleaving the proteolytically cleavable linker are more active or more highly expressed in a specific microenvironment (e.g., a disease microenvironment, such as a cancer microenvironment).

[0218] In embodiments of the multispecific antigen-binding constructs provided herein, the second antigen-binding domain is a masking domain that modulates the binding of the APP-binding domain to APP. In some embodiments of the multispecific antigen-binding constructs provided herein, the second antigen-binding domain is a masking domain that modulates the binding of CD47 to SIRPα.

[0219] In some embodiments, a first exemplary state of the binder may be a state in which the protein-hydrolyzable cleavable linker is intact, such that the associated masking domain can inhibit APP binding via the APP-binding domain. In some embodiments, a second exemplary state of the binder may be a state in which the protein-hydrolyzable cleavable linker has been cleaved to release the masking domain from the binder. In some embodiments, the first exemplary state is characterized in that the binder does not induce or does not significantly induce myeloid cell activity (e.g., phagocytosis of target cells) on 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) on target cells.

[0220] In some embodiments, the first exemplary state and the second exemplary state exhibit differential effects on myeloid cells and / or target cells (e.g., direct and / or indirect target cell killing, such as target cell phagocytosis) between activation conditions (e.g., activated microenvironment) and deactivation conditions (e.g., deactivated microenvironment). In some embodiments, the activation condition refers to an environment including a protease capable of cleaving the proteolytically cleaving linker of the binder disclosed herein. In some embodiments, the deactivation condition refers to an environment not including a protease capable of cleaving the proteolytically cleaving linker of the binder disclosed herein. In some embodiments, the binder does not enhance or significantly enhance myeloid cell activity and / or target cell killing (e.g., direct and / or indirect target cell killing, such as target cell phagocytosis) under deactivation conditions.

[0221] In some embodiments, the binder enhances (e.g., significantly enhances) myeloid cell activity and / or target cell killing (e.g., direct and / or indirect killing) under activation conditions. In some embodiments, the binding agent induces an enhancement or level of myeloid cell activity and / or target cell killing (e.g., direct and / or indirect killing) under activation conditions that is at least 10% greater than the enhancement or level of myeloid cell activity and / or target cell killing (e.g., direct and / or indirect killing) under deactivation conditions (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 100-fold, at least 1,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, at least 1,000,000-fold, or higher).

[0222] In some embodiments, the affinity of an app-binding domain not bound to a masking domain (e.g., under active conditions) to its target app is at least 20% greater than the affinity of an app-binding domain bound to a masking domain (e.g., under inactive conditions). In some embodiments, the affinity of an app-binding domain not bound to a masking domain (e.g., under active conditions) to its target app is at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 100 times, or at least 1000 times greater than the affinity of an app-binding domain bound to a masking domain (e.g., under inactive conditions).

[0223] In some embodiments, the affinity of an APP-binding domain not bound to a masking domain (e.g., under activation conditions) to its target APP may be equal to or less than about 200 nM. In some embodiments, the affinity of an APP-binding domain not bound to a masking domain (e.g., under activation conditions) to its target APP may be equal to or less than about 500 nM, about 450 nM, about 400 nM, about 350 nM, about 300 nM, about 250 nM, 200 nM, about 100 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, and about... 25 nM, approximately 20 nM, approximately 15 nM, approximately 10 nM, approximately 8 nM, approximately 7.5 nM, approximately 7 nM, approximately 6.5 nM, approximately 6 nM, approximately 5.5 nM, approximately 5 nM, approximately 4 nM, approximately 3 nM, approximately 2 nM, approximately 1 nM, approximately 500 pM, approximately 100 pM, approximately 60 pM, approximately 50 pM, approximately 20 pM, approximately 15 pM, approximately 10 pM, approximately 5 pM, approximately 2 pM or lower. In some implementations, the binding affinity is below any of the following values: about 500 nM, about 450 nM, about 400 nM, about 350 nM, about 300 nM, about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5 pM, or about 2 pM or lower, or any range therebetween, such as about 5 nM to about 35 nM. In some implementations, the affinity of an app-binding domain that is not bound to a masking domain (e.g., under activation conditions) for its target app may be equal to or less than about 1 × 10⁻⁶. -7 M, such as equal to or less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or lower. In some embodiments, affinity (K) can be determined using methods well known in the art. D ), such as by using surface plasmon resonance (SPR) technology in BIACORE® measuring instruments.

[0224] In some implementations, the affinity (K) of the APP binding domain to the masking domain (e.g., under inactive conditions) DThe affinity of the APP-binding domain to its target APP may be equal to or greater than approximately 200 nM. In some embodiments, the affinity of the APP-binding domain not bound to the masking domain (e.g., under activation conditions) to its target APP may be equal to or greater than 500 nM, approximately 450 nM, approximately 400 nM, approximately 350 nM, approximately 300 nM, approximately 250 nM, 200 nM, approximately 100 nM, approximately 50 nM, approximately 45 nM, approximately 40 nM, approximately 35 nM, approximately 30 nM, approximately 2... 5 nM, approximately 20 nM, approximately 15 nM, approximately 10 nM, approximately 8 nM, approximately 7.5 nM, approximately 7 nM, approximately 6.5 nM, approximately 6 nM, approximately 5.5 nM, approximately 5 nM, approximately 4 nM, approximately 3 nM, approximately 2 nM, approximately 1 nM, approximately 500 pM, approximately 100 pM, approximately 60 pM, approximately 50 pM, approximately 20 pM, approximately 15 pM, approximately 10 pM, approximately 5 pM, approximately 2 pM or higher. In some implementations, the binding affinity is higher than any of the following values: about 500 nM, about 450 nM, about 400 nM, about 350 nM, about 300 nM, 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5 pM, or about 2 pM or higher, or any range therebetween, such as about 5 nM to about 35 nM. In some implementations, the affinity of an app-binding domain that is not bound to a masking domain (e.g., under activation conditions) for its target app may be equal to or greater than about 1 × 10⁻⁶. -7 M, such as equal to or greater than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or lower. In some embodiments, affinity (K) can be determined using methods well known in the art. D ), such as by using surface plasmon resonance (SPR) technology in BIACORE® measuring instruments.

[0225] In some implementations, when the APP binding domain is bound to the masking domain, the affinity of the APP binding domain to its corresponding APP is reduced to about 1 or less, about 1 / 2, about 1 / 5, about 1 / 10, about 1 / 20, about 1 / 50, about 1 / 100, about 1 / 500, about 1 / 1000, about 1 / 500, about 1 / 1000, about 1 / 5000 or less, or any range therebetween, such as about 1 / 500 to about 1 / 1000.

[0226] In some implementations, the dissociation constant of the masking domain binding to the APP-binding domain is higher than the dissociation constant of the APP-binding domain binding to the corresponding APP. In some implementations, when the masking domain is cleaved from the binder by a protein-hydrolyzable cleavage linker, the masking domain does not interfere with or compete with the binding of the APP-binding domain to its APP.

[0227] In some embodiments, the masking domain of the binder covered by this disclosure may be an antibody or antibody fragment that binds to the APP-binding domain of the binder. In some embodiments, the masking domain of the binder covered by this disclosure may be a receptor protein that binds to the APP-binding domain of the binder. In some embodiments, the masking domain of the binder covered 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 masking domain of the binder covered by this disclosure may be a receptor ligand that binds to the APP-binding domain of the binder. In some embodiments, the masking domain of the binder covered 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 masking domain of the binder covered by this disclosure may be a small molecule that binds to the APP-binding domain of the binder. In some embodiments, the masking domain of the binder covered by this disclosure may be an aptamer that binds to the APP-binding domain of the binder.

[0228] In some embodiments, the masking domain binds to the APP-binding domain of a receptor protein. In some embodiments, the masking domain binds to the APP-binding domain of a fragment or domain of a receptor protein. In some embodiments, the masking domain binds to the APP-binding domain of a receptor ligand. In some embodiments, the masking domain binds to the APP-binding domain of a fragment or domain of a receptor ligand. In some embodiments, the masking domain binds to the APP-binding domain of an antibody or antibody fragment. In some embodiments, the masking domain binds to the APP-binding domain of a small molecule. In some embodiments, the masking domain binds to the APP-binding domain of an aptamer.

[0229] In some embodiments, the masking domain binds as an APP-binding domain of the SIRPα protein. In some embodiments, the masking domain binds as an APP-binding domain of a fragment or domain of the SIRPα protein. In some embodiments, the masking domain binds as an APP-binding domain of the SIGLEC10 protein. In some embodiments, the masking domain binds as an APP-binding domain of a fragment or domain of the SIGLEC10 protein. In some embodiments, the masking domain binds as an APP-binding domain of the PD-1 protein. In some embodiments, the masking domain binds as an APP-binding domain of a fragment or domain of the PD-1 protein. In some embodiments, the masking domain binds as an APP-binding domain of the LILRB1 protein. In some embodiments, the masking domain binds as an APP-binding domain of a fragment or domain of the LILRB1 protein.

[0230] In some embodiments, the masking domain is combined with an APP-binding domain, which may include at least one immunoglobulin heavy chain and / or at least one immunoglobulin light chain. In some embodiments, the masking domain is combined with an APP-binding domain, which may include at least one immunoglobulin heavy chain variable domain and / or at least one immunoglobulin light chain variable domain. In some embodiments, the masking domain is combined with an APP-binding domain, which may include at least one CDR1, CDR2, and CDR3 of an immunoglobulin heavy chain variable domain and / or at least one CDR1, CDR2, and CDR3 of an immunoglobulin light chain variable domain. In some implementations, the masking domain binding to the APP binding domain may be or include: the extracellular domain (ECD) of a cell surface-expressed protein, a mutant form (variant) of the ECD of a cell surface-expressed protein engineered to improve binding to the target, an intracellular antibody, a domain antibody, an antibody mimic, Zybody®, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd' fragment, an Fd fragment, an isolated CDR or a group thereof, a single-chain antibody, a single-chain Fv (scFv), or a disulfide-linked Fv. (sdFv), peptide-Fc fusions, single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof), camel antibodies, camel-derived antibodies, masking antibodies (e.g., Probody®), affybody, anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), single-chain or tandem biantibodies (TandAb®), VHH, Anticalin®, Nanobody® small antibodies, BiTE®, ankyrin repeats 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.

[0231] 1. SIRPα binding domain This document provides a SIRPα binding domain. In some embodiments, the SIRPα binding domain may be used as a masking domain in the provided construct. In the embodiments described herein, the SIRPα binding domain is a VHH-containing molecule that includes at least one VHH domain that specifically binds SIRPα. In some embodiments, the VHH domain binds human SIRPα. In some embodiments, the VHH domain binds one or both alleles of wild-type human SIRPα. In some embodiments, the VHH domain binds a variant SIRPα that contains one or more amino acid modifications compared to wild-type SIRPα. In some embodiments, the VHH domain binds a variant SIRPα that has a higher affinity for CD47 than wild-type SIRPα.

[0232] Wild-type and various engineered variants of SIRPα are known and can be used as the APP-binding domain in the binding constructs described herein, including any of those described in Section II.A. In some embodiments, the VHH domain binds the high-affinity variant CV1 SIRPα (described in Weiskopf K et al., Science. 2013; Ho CC et al., JBC. 2015; corresponding to the mutants V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I, based on amino acid numbers of SEQ ID NO: 103 or SEQ ID NO: 104). In some embodiments, the VHH domain binds the variant SIRPα, which has a higher affinity for CD47 than the wild-type SIRPα but a lower affinity than the CV1 SIRPα variant. In some embodiments, the VHH domain binds the variant SIRPα with the E54Q mutation. In some of the provided embodiments, the VHH domain is panreactive and capable of binding the wild-type SIRPα allele and one or more variants, including CV1 or a variant with the E54Q mutation. In some of the provided embodiments, the VHH domain binds SIRPα having the sequence shown in any of SEQ ID NO: 10, 27, 99, or 109. In some of the provided embodiments, the VHH domain binds SIRPα having the sequence shown in any of SEQ ID NO: 103, 104, 105, or 108.

[0233] In some implementations, the VHH domain is an antibody fragment, which is a single monomeric variable antibody domain capable of selectively binding to a specific antigen. The VHH domain (also known as a single-domain antibody) has a molecular weight of only 12-15 kDa, much lower than that of a typical antibody (150-160 kDa) composed of two heavy protein chains and two light chains, and even lower than that of the Fab fragment (approximately 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (approximately 25 kDa, two variable domains, one from the light chain and one from the heavy chain).

[0234] A single-domain antibody is an antibody whose complementation-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, naturally occurring antibodies lacking a light chain, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and non-antibody-derived single-domain scaffolds. Single-domain antibodies can 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 antibody used herein is a naturally occurring single-domain antibody, referred to as a heavy-chain antibody lacking a light chain. For clarity, this variable domain derived from naturally occurring heavy-chain antibodies lacking a light chain is referred to herein as a VHH to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can 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 naturally occurring heavy-chain antibodies lacking a light chain; such VHHs are within the scope of this disclosure.

[0235] Methods for screening VHH domains (including VHH-binding peptides) with desired specificity for SIRPα include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzymatic assays, flow cytometry, and other immune-mediated techniques known in the art.

[0236] The VHH domains provided in this paper include SIRPα (human-derived), such as any of those described below.

[0237] In some embodiments, the VHH domain binding to SIRPα may be derived from a non-human species and be humanized. Humanized antibodies (such as VHH-containing peptides) can be used as therapeutic molecules because they reduce or eliminate the human immune response to non-human antibodies, which can lead to an immune response to antibody therapeutics and reduce the effectiveness of the therapeutic. Typically, humanized antibodies contain one or more variable domains, wherein 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. Optionally, humanized antibodies may also contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or enhance antibody specificity or affinity.

[0238] Humanized antibodies and their preparation methods have been reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and have also been described, for example, in Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033; US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 (described as “surface remodeling”); Dall'Acqua et al., (2005) Methods 36:43-60 (describes “FR shuffling”); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describes a “guided selection” approach to FR shuffling).

[0239] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a “best fit” method (see, for example, Sims et al. (1993) J. Immunol. 151:2296); frame regions derived from the common sequence of human antibodies from specific subgroups of the heavy chain variable region (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature (somatic mutant) frame regions or human germline frame regions (see, for example, Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and frame regions derived from screening FR libraries (see, for example, Baca et al. (1997) J. Biol. Chem. 272: 10678-10684 and Rosok et al. (1996) J. Biol. Chem. 272: 10678-10684). Biol. Chem. 271:22611-22618). Typically, the FR region of a VHH is replaced with a human FR region to prepare a humanized VHH. In some embodiments, certain FR residues of the human FR are replaced to improve one or more properties of the humanized VHH. VHH domains with such replaced residues are still referred to herein as “humanized”.

[0240] This article provides a SIRPα-binding VHH domain (SIRPα-binding VHH domain or SIRPα VHH domain), wherein the VHH domain contains a VHH amino acid sequence selected from any of SEQ ID NO: 13-21 and 28-36, or CDR1, CDR2, and CDR3 contained in 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 of SEQ ID NO: 13-21 and 28-36. In some implementations, the SIRPα VHH domain provided herein includes: CDR1 as shown in any of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44 and 45; CDR2 as shown in any of SEQ ID NO: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60; and CDR3 as shown in any of SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73. The provided SIRPα VHH domain includes an amino acid sequence having any one of the amino acid sequences shown in SEQ ID NO: 13-21 and 28-36, or 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 NO: 13-21 and 28-36. In some embodiments, the SIRPα VHH domain has an amino acid sequence having any one of the amino acid sequences shown in SEQ ID NO: 13-21 and 28-36.

[0241] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 13, or CDR1, CDR2, and CDR3 contained in 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: 13. In some embodiments, the SIRPα VHH domain comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 13.

[0242] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 14, or CDR1, CDR2, and CDR3 contained in 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: 14. In some embodiments, the SIRPα VHH domain comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 14.

[0243] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 15, or CDR1, CDR2, and CDR3 contained in 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: 15. In some embodiments, the SIRPα VHH domain comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 15.

[0244] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 16, or CDR1, CDR2, and CDR3 contained in 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 comprises 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. In some embodiments, the SIRPα VHH domain comprises the amino acid sequence shown in SEQ ID NO: 16.

[0245] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 17, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 17.

[0246] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 18, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 18.

[0247] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 19, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 19.

[0248] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 20, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 20.

[0249] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 21, or CDR1, CDR2, and CDR3 contained in 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: 21. In some embodiments, the SIRPα VHH domain comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 21.

[0250] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 28, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 28.

[0251] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 29, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 29.

[0252] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 30, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 30.

[0253] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 31, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 31.

[0254] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 32, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 32.

[0255] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 33, or CDR1, CDR2, and CDR3 contained in 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: 33. In some embodiments, the SIRPα VHH domain comprises the amino acid sequence shown 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 shown in SEQ ID NO: 33. In some embodiments, the SIRPα VHH domain comprises the amino acid sequence shown in SEQ ID NO: 33.

[0256] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 34, or CDR1, CDR2, and CDR3 contained in 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: 34. In some embodiments, the SIRPα VHH domain comprises the amino acid sequence shown 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 shown in SEQ ID NO: 34. In some embodiments, the SIRPα VHH domain comprises the amino acid sequence shown in SEQ ID NO: 34.

[0257] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 35, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 35.

[0258] In some embodiments, the SIRPα VHH domain provided herein comprises the VHH domain shown in SEQ ID NO: 36, or CDR1, CDR2, and CDR3 contained in 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 comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 36.

[0259] In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 37, 46, and 61, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 38, 46, and 61, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 39, 47, and 62, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 40, 48, and 63, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 41, 49, and 64, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 37, 50, and 61, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 42, 51, and 65, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 43, 52, and 66, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 37, 53, and 67, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 44, 54, and 68, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 43, 55, and 63, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 40, 56, and 69, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 37, 57, and 70, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 40, 55, and 63, respectively.In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 41, 58, and 71, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 43, 59, and 72, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 37, 60, and 73, respectively. In some embodiments, the SIRPα VHH domain provided herein includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 45, 56, and 73, respectively.

[0260] 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 domain provided herein includes 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.

[0261] In some implementations, the SIRPα VHH domain is used as a masking domain in any of the provided multispecific antigen-binding constructs, such as to mask the CD47 APP binding domain.

[0262] In some embodiments, the SIRPα VHH domain is used as a component of a bispecific macrophage adaptor (BiME). In some embodiments, a bispecific molecule is provided comprising: a SIRPα VHH domain that binds to SIRPα and blocks the interaction between CD47 on one cell and SIRPα on a phagocyte; and a second antigen. Antibodies with bispecificity against both SIRPα and the second antigen are referred to as bispecific macrophage enhancement (BiME) antibodies. In some embodiments, such binding molecules can trigger phagocytosis and an immune response against target cells, such as tumor cells. In some embodiments, the bispecific binding molecule can bridge two closely adjacent cells (effector cell and target cell) such that other cell receptors and membrane components on either cell can interact, and myeloid effector cells can thereby trigger phagocytosis of the target cell.

[0263] In some embodiments, the bispecific antibody targets SIRPα and a second antigen. Therefore, in some cases, the subject 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 antigens as described in Table 2. Other exemplary second antigens are any cancer cell markers, such as 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 some cases, exemplary bispecific antibodies include the SIRPα VHH domain sequence (e.g., CDR) disclosed herein that provides specific binding to SIRPα, and a sequence (e.g., CDR) from an antibody that binds to a cancer cell marker. Examples of antibodies that provide specific binding to cancer cell markers include any antibody as described herein. In some embodiments, the antibody against the cancer marker is cetuximab (binding to EGFR), panitumumab (binding to EGFR), rituximab (binding to CD20), trastuzumab (binding to HER2), pertuzumab (binding to HER2), alemtuzumab (binding to CD52), or bentuximab (binding to CD30).

[0264] (i) Exemplary features The physical / chemical properties and / or biological activities of the SIRPα VHH domains presented herein can be identified, screened, or characterized using various known assays.

[0265] In some embodiments, the SIRPα VHH domain has one or more specific functional characteristics, such as binding properties, including binding to SIRPα (such as wild-type SIRPα and / or variant SIRPα). In some embodiments, the SIRPα VHH domain is capable of binding SIRPα with at least a certain affinity. In some embodiments, the provided SIRPα VHH domain binds to specific epitopes, 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 any of a variety of known methods. The binding affinity can be measured as K. D K A or EC 50 In some implementations, the affinity is determined by the equilibrium dissociation constant (K). D (This is indicated by ) In some implementations, affinity is determined by EC. 50 express.

[0266] Several methods are known for evaluating binding affinity and equilibrium dissociation constant (K). D ), equilibrium association constant (K) A EC 50 Binding rate (association rate constant; k) on or k a The unit is 1 / Ms or M. -1 s -1 ) and dissociation rate (dissociation rate constant; k off or k d The unit is 1 / s or s. -1 This involves 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 the binding molecule can be determined, for example, by using any of a variety of well-known binding assays. For instance, in some embodiments, a Carterra® LSA™ instrument can be used to determine the binding kinetics and constants of a complex between two proteins (e.g., an antibody or a fragment thereof and an antigen) using surface plasmon resonance (SPR) analysis (see, for example, Scatchard et al.). Ann. NY Acad. Sci. 51 :660, 1949; Wilson, Science 295 :2103, 2002; Wolff et al., Cancer Res . 53(2560, 1993). In one aspect, the SIRPα binding activity of the SIRPα VHH domain is tested, for example, by known methods. In some embodiments, the binding of the SIRPα VHH domain to an antigen (such as SIRPα, such as wild-type SIRPα or variant SIRPα) is assessed by methods known in the art, such as ELISA, Western blotting, flow cytometry assays, and / or surface plasmon resonance (SPR).

[0267] In some implementations, the SIRPα VHH domain and the equilibrium dissociation constant (K) of SIRPα are... D The values ​​are: approximately 0.1 nM to approximately 10 µM, approximately 0.1 nM to approximately 50 nM, approximately 0.1 nM to approximately 40 nM, approximately 0.1 nM to approximately 30 nM, approximately 0.1 nM to approximately 20 nM, approximately 0.1 nM to approximately 10 nM, approximately 0.1 nM to approximately 1 nM, approximately 1 nM to approximately 500 nM, approximately 1 nM to approximately 50 nM, or about 1 nM to about 40 nM, or about 1 nM to about 30 nM, or about 1 nM to about 20 nM, or about 1 nM to about 10 nM, or about 10 nM to about 500 nM, or about 10 nM to about 50 nM, or about 10 nM to about 40 nM, or about 10 nM to about 30 nM, or about 10 nM to about 20 nM. In some embodiments, the binding affinity (EC) of the VHH domain to SIRPα is... 50 ) and / or equilibrium dissociation constant (K D The range is or is about or less than about 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 a range defined by any of the aforementioned values.

[0268] In some embodiments, the SIRPα VHH domain binds SIRPα with a sub-nanomolar binding affinity, for example, a binding affinity of less than or below about 1 nM, such as less than or below about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In some embodiments, the equilibrium dissociation constant K of the binding molecule (e.g., the SIRPα VHH domain) with SIRPα (such as wild-type or variant SIRPα) is... DThis is or is 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. In some embodiments, the equilibrium dissociation constant K of the binding molecule (e.g., the SIRPα VHH domain) with SIRPα is... D The value is or is about or less than about 10 μM, 5 μM, 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 lower, or a range defined by any of the foregoing values.

[0269] In some embodiments, the provided SIRPα VHH domain binds to SIRPα, and its binding affinity allows for spontaneous release of the SIRPα VHH domain from SIRPα after cleavage of the protease-cleavable linker. In some embodiments, to enhance the antiphagocytic activity of the provided multispecific binding construct, the provided SIRPα VHH domain... k The off value cannot be too low and needs to be higher than that of the construct's wild-type SIRPα or variant SIRPα against wild-type human CD47 (such as cell surface-expressed CD47). Furthermore, the binding rate of the construct's wild-type SIRPα or variant SIRPα to wild-type human CD47 (such as cell surface-expressed CD47) should be faster than the rebinding rate of cleaved and dissociated masking agents, allowing the SIRPαVHH domain to... k The on value should be lower than that of wild-type SIRPα or variant SIRPα antigen. In some embodiments, the SIRPα VHH domain binds to the wild-type SIRPα or variant SIRPα of the construct with a lower affinity than the wild-type SIRPα or variant SIRPα of the construct binds to wild-type human CD47 (such as CD47 expressed on the cell surface), and therefore has a higher dissociation constant (K). D In some implementations, the SIRPα VHH domain binds to the dissociation constant (K0) of wild-type SIRPα or its variants. D SIRPα or its variants are the K-type receptors for wild-type CD47 (such as CD47 expressed on the cell surface). DAt least 2, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times. In some implementations, the K of the SIRPα VHH domain... D Not less than 1 nM. In such embodiments, after the protease-cleavable linker is cleaved (where the SIRPα VHH domain is linked to SIRPα via the protease-cleavable linker), the SIRPα VHH domain is released from SIRPα. In such instances, SIRPα no longer binds to the SIRPα VHH domain, and SIRPα can interact with other binding partners (such as CD47).

[0270] C. Protein-hydrolyzable cutting connector This disclosure particularly provides binding agents comprising a masking domain that associates with a protein-hydrolyzable cleavable linker. In some embodiments, the protein-hydrolyzable linker is present in a polypeptide comprising multiple domains provided herein (e.g., comprising at least an APP-binding domain and a masking domain). In some embodiments, the masking domain is positioned such that cleavage of the protein-hydrolyzable linker separates the masking domain from at least one other domain or all other domains present in the polypeptide and / or the binding agent. In this way, the masking domain substantially inhibits the binding of the APP-binding domain to the APP target until after the protein-hydrolyzable peptide linker has been cleaved in the desired environment.

[0271] In some embodiments, the protein-hydrolyzable cleavage adapter includes cleavage sites, such as protease cleavage sites that are recognized by and can be cleaved by proteases. The protein-hydrolyzable cleavage adapter contains an amino acid sequence that can be used as a substrate for proteases, such as extracellular proteases.

[0272] In some embodiments, the hydrolyzable cleavable adapter can be cleaved by human and / or biologically relevant proteases (e.g., proteases expressed by one or more cells of the human body and / or in one or more tissues). In some embodiments, the hydrolyzable cleavable adapter can be cleaved by extracellular proteases. In some embodiments, the hydrolyzable cleavable adapter can be cleaved by cell surface proteases. In some embodiments, the hydrolyzable cleavable adapter can be cleaved by intracellular proteases. In some embodiments, the hydrolyzable cleavable adapter can be cleaved by aminopeptidases. In some embodiments, the hydrolyzable cleavable adapter can be cleaved by aspartic proteases. In some embodiments, the hydrolyzable cleavable adapter can be cleaved by metalloproteinases. In some embodiments, the hydrolyzable cleavable adapter can be cleaved by cysteine ​​proteases. In some embodiments, the hydrolyzable cleavable adapter can be cleaved by serine proteases. In some embodiments, the hydrolyzable cleavable adapter can be cleaved by threonine proteases.

[0273] In some embodiments, the proteolytically cleavable linker may be cleaved by a human protease selected from the following and / or contains a motif cleaved by a human protease selected from the following: ABHD12 (containing exfoliase domain protein 12), ABHD12B (containing exfoliase domain protein 12B), ABHD13 (containing exfoliase domain protein 13), ABHD17A (sequence similarity family 108 member A1), ABHD17B (sequence similarity family 108 member B1), ABHD17C (sequence similarity family 108 member C1), ABHD4 (containing exfoliase domain protein 4), ABHD5 (CGI-58), ACE (angiotensin-converting enzyme 1), ACE2 (angiotensin-converting enzyme 2), ACE3P (angiotensin-converting enzyme 3), ACR (acromone protein), ACY1 (aminoacylase), ACY3 (Aspartic acid acylase-3), ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM1A (ADAM1a), ADAM2 (ADAM2 / fertilin-b), ADAM20, ADAM21, ADAM22, ADAM23, ADAM25 (testisin 2), ADAM28, ADAM29, ADAM30, ADAM32, ADAM33, ADAM3B (ADAM3B), ADAM4, ADAM4B (ADAM4B), ADAM5, ADAM6, ADAM7, ADAM8, ADAM9, ADAMDEC1 (DECYSIN), ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ADAMTS5 (ADAMTS5 / 11), ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADGB (calpain 7-like protein), AEBP1 (adipocyte-enh binding protein 1), AFG3L1P (Afg3-like protein 1), AFG3L2 (Afg3-like protein 2), AGA (glycosylasparaginase), 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-acetylglucosamine transferase subunit), AMZ1 (archaeal metalloproteinase-1), AMZ2(Archaeogenic metalloproteinase-2), ANPEP (aminopeptidase N), AOPEP (aminopeptidase O), APEH (acylaminoacylpeptidase), ASAH1 (acid ceramidinase), ASPA (aspartic acid acylase), ASPRV1 (DDI-associated protease), ASRGL1 (glycosyl asparaginase-2), ASTL (ovalbuminase), ATG4A (autophagosome-2), ATG4B (autophagosome-1), ATG4C (autophagosome-3), ATG4D (autophagosome-4), ATXN3 (conjugin-3), ATXN3L (conjugin-3-like protein), AZU1 (azurin), BACE1 (β-secretase 1), BACE2 (β-secretase 2), BAP1 (ubiquitin C-terminal hydrolase BAP1), BLMH (bleomycin hydrolase), BMP1 (procollagen C-protease), BRCC3 (containing BRCC36 / BRCA2 complex subunit 3), C1R (Complement component C1ra), C1RL (Complement C1r homolog), C1S (Complement component C1sa), C2 (Complement component 2), CAD (Dihydroorotase), CAPN1 (Calcenase 1), CAPN10 (Calcenase 10), CAPN11 (Calcenase 11), CAPN12 (Calcenase 12), CAPN13 (Calcenase 13), CAPN14 (Calcenase 14), CAPN15 (Calcenase 15 / Solh protein), CAPN2 (Calcenase 2), CAPN3 (Calcenase 3), CAPN5 (Calcenase 5), CAPN6 (Calcenase 6), CAPN7 (Calcenase 7), CAPN8 (Calcenase 8), CAPN9 (Calcenase 9), CARD8 (Caspase recruitment domain family member 8), CASP1 (Caspase-1), CASP10 (Caspase-10), CASP12 (Caspase-12), CASP14 (Caspase-14), CASP16P (Caspase-14-like protein), CASP1P2 (homogeneous protein 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 type B), CELA3A (pancreatic endopeptidase E (A)), CELA3B (pancreatic endopeptidase E (B)), CFBComplement Factor B, CFD (Complement Factor D), CFI (Complement Factor I), CFLAR (Casper / FLIP), CLPP (Endopeptidase Clp), CMA1 (Chymotrypsin), CNDP1 (Carnosine Dipeptidase 1), CNDP2 (Carnosine Dipeptidase 2), COPS5 (CSN5 / JAB1), COPS6 (COPS6), CORIN (Corynexin), 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 (Carboxypeptidase D), CPE (Carboxypeptidase E), CPM (Carboxypeptidase M), CPN1 (Carboxypeptidase N), CPO (Carboxypeptidase O), CPQ (Plasma Glu-Carboxypeptidase), CPVL (Egg Yolk Carboxypeptidase-L), CPXM1 (Carboxypeptidase X1), CPXM2 (Carboxypeptidase X2), CPZ (Carboxypeptidase Z), CRMP1 (Dihydropyrimidine enzyme-associated protein 1), CTRB1 (Cymotrypsin B), CTRC (Cymotrypsin C), CTRL (Esophageal chymotrypsin), CTSA (Lysosomal carboxypeptidase A), CYLD (Cymotrypsin), CYMP (Cymotrypsin), 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 (Desmearylisopeptidase 1), DESI2 (Desmearylisopeptidase 2), DHH (Desert hedgehog protein), DNPEP (Aspartate aminopeptidase), 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 (dihydropyrimidine enzyme), DPYSL2 (dihydropyrimidine enzyme-associated protein 2), DPYSL3 (dihydropyrimidine enzyme-associated protein 3), DPYSL4 (dihydropyrimidine enzyme-associated protein 4), DPYSL5 (dihydropyrimidine enzyme-associated protein 5), ECE1 (endothelin-converting enzyme 1), ECE2 (endothelin-converting enzyme 2), ECE1 (DINE peptidase), ECT2L (epithelial cell transformation sequence 2 oncogene-like protein), EIF3F (eukaryotic translation initiation F3SF), EIF3H (eukaryotic translation initiation F3SH), ELANE(Neutral elastase), ENPEP (aminopeptidase A), EPHX1 (epoxyhydrolase), EPHX4 (epoxyhydrolase-associated protein), ERAP1 (aminopeptidase PILS), ERAP2 (aminopeptidase MAMS / L-RAP), ERMP1 (endoplasmic reticulum metallopeptidase 1), ESPL1 (separating enzyme), 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 / ZMPSTE24), FACE2 (FACE-2 / RCE1), FAM111A (sequence similarity family 111 A), FAM111B (sequence similarity family 111 B), FAP (fibroblast activator protein), FOLH1 (glutamate carboxypeptidase II), FREM1 (signal enzyme-like protein 1), FURIN (furin protease), GFPT1 (Gln-fructose-6-P transaminase 1), GFPT2 (Gln-fructose-6-P transaminase 2), GFPT3 (Gln-fructose-6-P transaminase 3), GGH (γ-glutamyl hydrolase), GGT1 (γ-glutamyl transferase 1), GGT2 (γ-glutamyl transferase 2), GGT6 (γ-glutamyl transferase 6), GGT7 (γ-glutamyl transferase-like protein 3), GGTLC1 (γ-glutamyl transferase 5), GGTLC2 (γ-glutamyl transferase m-3), GZMA (granulase A), GZMB (granulase B), GZMH (granulase H), GZMK (granulase K), GZMM (granulase M), HABP2 (hyaluronic acid-binding serine protease), HATL2 (HAT-like protein 2), HATL3 (HAT-like protein 3), HGF (hepatocyte growth factor), HGFAC (HGF activator), HM13 (Progeria homolog 3 / SPP), HP (haptoglobin-1), HPN (hepatic serine protease), HPR (haptoglobin-associated protein), HSP90AA1 (heat shock 90kDa protein 1 α), HSP90AB1 (heat shock 90kDa protein 1 β), HSP90B1 (heat shock protein 90kDa β (Grp94) member 1 / tumor rejection antigen (gp96)), HTRA1 (osteoblast serine protease), HTRA2 (HTRA2), HTRA3 (HTRA3), HTRA4 (HTRA4), IDE (insulin-degrading enzyme), IHH (Indian hedgehog protein), IMMP1L (mitochondrial signal peptidase), IMMP2L (mitochondrial inner membrane protease 2), INPP5E (mitochondrial processing protease), JOSD1Josephine protein-1, JOSD2 (Josephine protein-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 (β-lactamase), LAP3 (leucylaminopeptidase), LGMN (leucine), LMLN (leishmaniazide-2), LNPEP (leucyl-cysteine-like 1), LOC440434 (cytoplasmic alanine aminopeptidase-like protein 1), LONP1 (PIM1 endopeptidase), LONP2 (PIM2 endopeptidase), LPA (apolipoprotein), LTA4H (leukotriene A4 hydrolase), LTF (lactoferrin), LVRN (aminopeptidase Q), MALT1 (paracysteine), MASP1 (MASP1 / 3), MASP2 (MASP2), MASTIN (mast cell protease), MBTPS1 (site 1 protease), MBTPS2 (S2P protease), MEP1A (Hypnotin α subunit), MEP1B (Hypnotin β subunit), MEST (Mesodermal-specific transcript), METAP1 (Methionyl aminopeptidase I), METAP1D (MAP1D Methionine Aminopeptidase 1D), METAP2 (Methionyl aminopeptidase II), MIPEP (Mitochondrial intermediate peptidase), MME (Enkephalin), MMEL1 (Enkephalin-2), MMP1 (Collagenase 1), MMP10 (Lysolysin 2), MMP11 (Lysolysin 3), MMP12 (Macrophage elastase), MMP13 (Collagenase 3), MMP14 (MT1-MMP), MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MMP), MMP19 (MMP19), MMP2 (Gelatinase A), MMP20 (Ameliolysin), MMP21 (MMP21), MMP23AMMP23A, MMP23B, MMP24 (MT5-MMP), MMP25 (MT6-MMP), MMP26 (stromallosing factor-2), MMP27 (MMP27), MMP28 (epidermolysin), MMP3 (stromallosing factor-1), MMP7 (stromallosing factor), 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 (napeptidase A), NAPSB (napeptidase B), NLN (neurolysin), NLRP1 (NLRP1 autocleavage protein), NPEPL1 (aminopeptidase-like protein 1), NPEPPS (Cytoplasmic alanine aminopeptidase), NRD1 (Nardilixin), NRIP2 (Nuclear receptor interaction protein 2), NRIP3 (Nuclear receptor interaction protein 3), NSMF (Nasal embryonic LHRH factor), NUP98 (Nuclear porin 98), NYNRIN (containing NYN domain and retroviral integrase), OMA1 (OMA1), OSGEP (O-sialic acid glycoprotein endopeptidase), OSGEPL1 (O-sialic acid glycoprotein endopeptidase-like protein 1), OTUB1 (Otuban protein-1), OTUB2 (Otuban protein-2), OTUD1 (containing OTU domain-1), OTUD3 (containing OTU domain-3), OTUD4 (Hin-1 / containing OTU domain-4), OTUD5 (containing OTU domain-5), OTUD6A (containing OTU domain-6A), OTUD6B (containing OTU domain-6B), OTUD7A (Cezanne protease-2), OTUD7B (Cezanne protease / containing OTU domain 7B), OVCH1 (oochymase-like protein), OVCH2 (ovulatory protein-like / oochymase-2), PA2G4 (proliferation-associated protein 1), PAMR1 (protein C-like protein), PAN2 (USP52), PAPPA (coronavirus-1), PAPPA2 (coronavirus-2), PARK7 (DJ-1), PARL (progerin-associated rhomboid protein), PCSK1 (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-proline dipeptidase), 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 (additional metalloproteinase 1), PLAT (t-plasminogen activator), PLAU (u-plasminogen activator), PLG (plasminogen), PM20D2 (PM20D2 peptidase), PMPCB (mitochondrial processing peptidase β-subunit), PPAT (Gln-PRPP amidotransferase), PPNX (Ppnx), PRCP (lysosomal proline-X C-peptidase), PREP (prolyl oligopeptidase), PREPL (prolyl oligopeptidase-like protein), PROC (protein C), PROZ (protein Z), PRPF8 PRSS1 (Catonic trypsin), PRSS12 (Neurotrypsin), PRSS16 (Thymus-specific serine peptidase), PRSS2 (Anionic trypsin(II)), PRSS21 (Testisin), PRSS22 (Brain serine protease 2), PRSS23 (Umbilical vein protease), PRSS27 (Malaprocin), PRSS29P (Implantation serine protease 2), PRSS3 (Intermediate trypsin), PRSS30P (Intestinal serine protease 1), PRSS33 (Treatin homolog 2 / EOS), PRSS35 (Similar to SPUVE), PRSS36 (Polyserinase-2), PRSS37 (Trypsin X2), PRSS38 (Malaprocin 2), PRSS3P2 (Trypsin C), PRSS41 (Treatin homolog 3), PRSS42 (Testisin serine protease 2), PRSS45 (Testisin serine protease 5), PRSS48 (Epidermal-specific SP-like protein), PRSS50 (Testis-specific protein Tsp50), PRSS53 (polyserine enzyme-3), PRSS54 (plasma kallikrein-like protein 4), PRSS55 (plasma kallikrein-like protein 2), PRSS56 (serine protease 56), PRSS57 (complement factor D-like protein), PRSS8 (prostaglandin), PRTN3 (protease 3), PSEN1 (presenilin 1), PSEN2 (presenilin 2), PSMA1 (proteasome α1 subunit), PSMA2 (proteasome α2 subunit), PSMA3 (proteasome α3 subunit), PSMA4 (proteasome α4 subunit), PSMA5 (proteasome α...PSMB1 (proteasome α-6 subunit), PSMB6 (proteasome α-7 subunit), PSMB7 (proteasome α-8 subunit), PSMB1 (proteasome β-1 subunit), PSMB10 (proteasome catalytic subunit 2i), PSMB11 (proteasome β-subunit LMP7-like protein), PSMB2 (proteasome β-2 subunit), PSMB3 (proteasome β-3 subunit), PSMB4 (proteasome β-4 subunit), PSMB5 (proteasome catalytic subunit 3), PSMB6 (proteasome catalytic subunit 1), PSMB7 (proteasome catalytic subunit 2), PSMB8 (proteasome catalytic subunit 3i), PSMB9 (proteasome catalytic subunit 1i), PSMD14 (POH1 / PSMD14), PSMD7 (PSMD7), QPCT (glutamine acyl cyclase), QPCTL (glutamine acyl cyclase 2), RBP3 (retinol-binding protein 3), RELN (complexin), REN (Renin), RHBDD1 (containing rhomboid domain 1), RHBDD2 (containing rhomboid domain 2), RHBDF1 (rhomboid protein 5 homolog 1), RHBDF2 (rhomboid protein 5 homolog 2), RHBDL1 (rhomboid protein-like protein 1), RHBDL2 (rhomboid vein-like protein 2), RHBDL3 (rhomboid vein-like protein 3), RNPEP (aminopeptidase B), RNPEPL1 (aminopeptidase B-like protein 1), SCPEP1 (serine carboxypeptidase 1), SCRN1 (secretin-1), SCRN2 (secretin-2), SCRN3 (secretin-3), SEC11A (signal enzyme 18kDa fraction), SEC11C (signal enzyme 21kDa fraction), SENP1 (sentinel protein / SUMO protease 1), SENP2 (sentinel protein / SUMO protease 2), SENP3 (sentinel protein / SUMO protease 3), SENP5 (sentinel protein / SUMO protease 5), SENP6 (sentinel protein / SUMO protease 6), SENP7 (Sentinel protein / SUMO protease 7), SENP8 (Sentinel protein / SUMO protease 8), SHH (Sound hedgehog protein), SPG7 (Paraplegic protein), SPPL2A (Progerin homolog 5), SPPL2B (Progerin homolog 4 / SPPL2B), SPPL2C (Progerin homolog 2), SPPL3 (Progerin homolog 1 / SPPL3), SPRTN (SprT-like N-terminal domain), ST14 (Matrix protease), STAMBP (AMSH / STAMBP), STAMBPPL1 (AMSH-LP / STAMBPL1), SUPT16H (Ty16 inhibitory factor homologs), TAF2 (TBP-associated factor 2), TASP1 (threonine aspartate), TESP2 (TESP2), TESP3 (TESP3), TESSP3 (testisine serine protease 3), TESSP4 (testisine serine protease 4), TESSP6 (testisine serine protease 6), TFR2 (transferrin receptor 2 protein), TFRC (transferrin receptor protein), THOP1 (thioctin oligopeptidase), TINAG (tubule-interstitial nephritis antigen), TINAGL1 (TINAG-associated protein), TLL1 (mammalian tolloid-like 1 protein), TLL2 (mammalian tolloid-like 2 protein), TMPRSS11A (TMPRSS11A), TMPRSS11B (HAT-like protein 5), TMPRSS11D (airway trypsin-like protease), TMPRSS11E (DESC1 protease), TMPRSS11F (HAT-like protein 4), TMPRSS12 (HAT-associated protease), TMPRSS13 (Membrane-type chimeric serine protease), TMPRSS15 (intestinal peptidase), TMPRSS2 (epithelial protease), TMPRSS3 (transmembrane serine protease 3), TMPRSS4 (transmembrane serine protease 4), TMPRSS5 (spinal protease), TMPRSS6 (matrix protease-2), TMPRSS7 (matrix protease-3), TMPRSS9 (polyserine protease-I), TNFAIP3 (A20, TNFα-inducible protein 3), TPP1 (tripeptidyl peptidase I), TPP2 (tripeptidyl peptidase II), TPSAB1 (trypsin α / β 1), TPSB2 (trypsin β 2), TPSD1 (trypsin δ 1), TPSG1 (trypsin γ 1), TRAP1 (heat shock protein 75), TRHDE (TRH-degrading extracellular enzyme), TRY10 (trypsin 10), TRY15 (trypsin 15), TTC28 (HetF-like protein), TTR (transthyretin), TYSND1 (Similar to Arabidopsis serine protease), 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), USP15(USP15), USP16 (USP16), USP17L2 (USP17-like protein), 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), USP32 (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 protein 1), VCPIP1 (VCP(p97) / p47 interacting protein), XPNPEP1 (aminopeptidase P1), XPNPEP2 (X-prolyl aminopeptidase 2), XPNPEP3 (aminopeptidase P homolog), XRCC6BP1 (ATP23 peptidase), YME1L1 (YME1-like protein 1), YOD1 (OTUD2 / YOD1), ZC3H12A (containing CCCH type zinc finger 12A), ZC3H12B (containing CCCH type zinc finger 12B), ZC3H12C (containing CCCH type zinc finger 12C), ZC3H12D (containing CCCH type zinc finger 12D), ZRANB1 (TRAF binding protein domain) and / or ZUP1 (containing zinc finger ubiquitin peptidase 1).

[0274] In some embodiments, the proteolytically cleavable adapter may be cleaved by a human protease selected from the following and / or contain a motif cleaved by a human protease selected from the following: renin, cathepsin D, cathepsin E, pepsin C or aspartic protease A, matrix metalloproteinase (MMP), matrix protease, urokinase-type plasminogen activator (uPA), integrin metalloproteinase (ADAM), integrin metalloproteinase containing a platelet-reactive protein motif (ADAMTS), podocyte oleoresin, urokinase, or hepatic serine.

[0275] Numerous representative examples of protein-hydrolyzable cleavage linker sequences are known in the art, and assays for determining protein sequence cleavage by proteases are also well known in the art (see, for example, U.S. Patent 10,259,845 and U.S. Patent Publication 2020 / 0115461).

[0276] In some embodiments, the proteolytically cleavable linker includes a protease cleavage site, which is a tumor-associated protease cleavage site recognized by the protease, the expression of which is specific to or upregulated in tumor cells or their tumor cell environment. The cleavable linker can be selected based on the protease produced by tumors located near cells expressing the target and / or by tumors co-localized in tissues with the desired target of the multispecific polypeptide construct. Elevated levels of proteases with known substrates have been reported in various cancers (e.g., solid tumors). See, for example, La Rocca et al., (2004) British J. of Cancer 90(7): 1414-1421.

[0277] In some embodiments, the protease cleavage site is a cleavage site recognized by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20. , ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1 , CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTR1, CTSA, CTSW, CTSB, C TSC, 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, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, H TRA3, HTRA4, KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5 , PCSK6, MME, MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13,MMP14, ST14, TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRS S2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1 and TPSG1. ,

[0278] 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.

[0279] In the embodiments, the protease cleavage site is a matrix metalloproteinase (MMP) cleavage site, a metalloproteinase cleavage site containing an integrin-containing metalloproteinase domain (ADAM), 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 matrix protease protease cleavage site (ST14), or a legume protease cleavage site. In the embodiments, the matrix metalloproteinase (MMP) cleavage site is an MMP9 cleavage site, an MMP13 cleavage site, or an MMP2 cleavage site. In the embodiments, the metalloproteinase cleavage site containing an integrin-containing metalloproteinase domain (ADAM) is an ADAM9 metalloproteinase cleavage site, an ADAM10 metalloproteinase cleavage site, or an ADAM17 metalloproteinase cleavage site.

[0280] In some embodiments, the protein-hydrolyzable cleavable linker is a cleavable peptide. In some embodiments, the cleavable peptide is a pentamelider (i.e., a peptide of 5 amino acids in length), a hexamer (i.e., a peptide of 6 amino acids in length), a heptamer (i.e., a peptide of 7 amino acids in length), an octamer (i.e., a peptide of 8 amino acids in length), a nonamelider (i.e., a peptide of 9 amino acids in length), a decamer (i.e., a peptide of 10 amino acids in length), an 11-mer (i.e., a peptide of 11 amino acids in length), a 12-mer (i.e., a peptide of 12 amino acids in length), a 13-mer (i.e., a peptide of 13 amino acids in length), a 14-mer (i.e., a peptide of 14 amino acids in length), a 15-mer (i.e., a peptide of 15 amino acids in length), a 16-mer (i.e., a peptide of 16 amino acids in length), a 17-mer (i.e., a peptide of 17 amino acids in length), or an octamer (i.e., a peptide of 18 amino acids in length).

[0281] In some embodiments, the cleavable adapter contains a substrate recognition site or cleavage site for a specific protease, which is a sequence recognized by the active site of the protease and cleaved by the protease. Typically, for example, for serine proteases, the cleavage sequence consists of amino acids P1-P4 and P1′-P4′ in the substrate, with cleavage occurring after the P1 position. Typically, the cleavage sequence of a serine protease is six residues long to match the extended substrate specificity of various proteases, but depending on the protease, the sequence can be longer or shorter. Typically, the cleavable adapter includes a P1-P1′ cleavable bond sequence recognized by the protease. In some aspects, the cleavable adapter is engineered to introduce peptide bonds that can be cleaved by a specific protease, for example, by introducing a substrate recognition site sequence or cleavage sequence of the protease.

[0282] In some embodiments, the protease is granzyme B, matrix protease, or MMP (such as MMP-2). In some embodiments, the cleavable adapter comprises 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.

[0283] In some embodiments, the cleavable linker comprises an amino acid that serves as a substrate for granzyme B. In some embodiments, the granzyme B cleavable linker contains an amino acid sequence having the general formula P4 P3 P2 P1 ↓ P1' (SEQ ID NO: 214), wherein P4 is amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino acid D or E; and P1' is amino acid I, L, Y, M, F, V, T, S, G, or A. In some embodiments, the granzyme B cleavable linker contains an amino acid sequence having the general formula P4 P3 P2 P1 ↓ P1' (SEQ ID NO: 260), wherein 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.

[0284] In some embodiments, the substrate of granzyme B comprises 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 comprises the amino acid sequence IEPDI (SEQ ID NO: 218), LEPDG (SEQ ID NO: 219, LEADT (SEQ ID NO: 220), IEPG (SEQ ID NO: 221), IEPV (SEQ ID NO: 222), IEPDS (SEQ ID NO: 223), IEPT (SEQ ID NO: 224), IEPDP (SEQ ID NO: 225), LEPDG (SEQ ID NO: 226), or LEADG (SEQ ID NO: 227).

[0285] In some embodiments, the cleavable adapter comprises an amino acid that serves as a substrate for the matrix protease. In some embodiments, the cleavable adapter comprises the sequence P4QAR↓(A / V) (SEQ ID NO: 228), where P4 is any amino acid. In some embodiments, the cleavable adapter comprises the sequence RQAR(A / V) (SEQ ID NO: 229). In some embodiments, the substrate for the matrix protease comprises the amino acid sequence RQAR (SEQ ID NO: 230). In some embodiments, the cleavable adapter comprises the amino acid sequence RQARV (SEQ ID NO: 231).

[0286] In some embodiments, the cleavable linker comprises an amino acid that serves as a substrate 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 P3P2 P1 ↓ 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 P3 P2 P1 ↓ 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 for the MMP comprises the amino acid sequence PAGL (SEQ ID NO: 233).

[0287] In some embodiments, the cleavable adapter comprises a combination of an amino acid sequence serving as a substrate for granzyme B and an amino acid sequence serving as a substrate for a matrix protease. In some embodiments, the cleavable adapter comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 215) and the amino acid sequence RQAR (SEQ ID NO: 230).

[0288] In some embodiments, the cleavable adapter comprises a combination of an amino acid sequence serving as a substrate for granzyme B and an amino acid sequence serving as a substrate for MMP. In some embodiments, the cleavable adapter comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 215) and the amino acid sequence PAGL (SEQ ID NO: 233).

[0289] In some embodiments, the cleavable adapter comprises a combination of an amino acid sequence serving as a substrate for a matrix protease and an amino acid sequence serving as a substrate for an MMP. In some embodiments, the cleavable adapter comprises a combination of the amino acid sequence RQAR (SEQ ID NO: 230) and the amino acid sequence PAGL (SEQ ID NO: 233).

[0290] In some embodiments, the cleavable adapter comprises a combination of amino acid sequences as substrates of granzyme B, substrates of matrix proteases, and substrates of MMPs. In some embodiments, the cleavable adapter comprises a combination of amino acid sequences as substrates of granzyme B and substrates of MMPs. In some embodiments, the cleavable adapter comprises a combination of the amino acid sequences LEAD (SEQ ID NO: 215), RQAR (SEQ ID NO: 230), and PAGL (SEQ ID NO: 233).

[0291] Cleavable connectors may include any known connector. Examples of cleavable connectors are described in Be'liveau et al. (2009) FEBS Journal, 276; U.S. Publication Applications Nos. US20160194399, US20150079088, US20170204139, US20160289324, US20160122425, US20150087810, and US20170081397; and U.S. Patent No. US9644016. Non-limiting cleavable peptide sequences are those in Table 1 of U.S. Patent No. 11,053,294, which is incorporated herein by reference in its entirety.

[0292] In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of: 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), GSLEADTGS (SEQ ID NO: 245), GGSGGGGIEPDGGGSGGS (SEQ ID NO: 246), GGSGGGIEPDVGGSGGS (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), GGGEGGGGSGGSGGGS (SEQ ID NO: 252), GSSAGSEAGGSGQAGVGS (SEQ ID NO: 253), GGSGGGLEAEGSGGGGS (SEQ ID NO: 254),GGSGGGGIEPDPGGSGGS (SEQ ID NO: 255), TGGGSGGGIEPDIGGSGGS (SEQ ID NO: 256).

[0293] In some embodiments, the protein-hydrolyzable cleavage linker is the cleavable peptide VHMPLGFLGPRQARVVN (SEQ ID NO: 22). In some embodiments, the protein-hydrolyzable cleavage linker is the cleavable peptide ISSGLLSGRSDNH (SEQ ID NO: 12).

[0294] In some embodiments, additional adapter sequences may be present at the N-terminus and / or C-terminus of the protease-cleavable adapter. In some embodiments, additional adapter sequences are present at the N-terminus of the protease-cleavable adapter. In some embodiments, additional adapter sequences are present at the C-terminus of the protease-cleavable adapter. In some embodiments, additional adapter sequences are present at both the N-terminus and C-terminus of the protease-cleavable adapter. The additional one or more adapter sequences are typically flexible adapter sequences. Particularly suitable adapter sequences primarily comprise amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, the adapter may contain at least 75% (based on the total number of residues present in the peptide adapter), such as at least 80%, at least 85%, or at least 90%, of amino acid residues selected from Gly, Ser, Ala, and Thr. The adapter may also consist only of Gly, Ser, Ala, and / or Thr residues. In some aspects, suitable peptide adapters typically contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, the peptide adapter contains only glycine residues. In some embodiments, the peptide linker contains only glycine and serine residues. In some embodiments, these linkers consist primarily of the amino acids glycine and serine, referred to herein as GS linkers. In some embodiments, the linker contains (GGS)n, where n is 1 to 5, such as 1 to 3. In a particular embodiment, the linker contains the sequence (GGGGS)n (SEQ ID NO: 259), where n is 1 to 5, such as 1 to 3. The linker may comprise a combination of any of the above, such as 2, 3, 4, or 5 repeats of GS, GGS, and / or GGGGS, and the linkers may be combined. In some embodiments, the length of such linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids. In some embodiments, the linker is (in single-letter amino acid codes): GGS or GGGGS (SEQ ID NO: 11), GGGGSGGGGS (SEQ ID NO: 9), and GGGGSGGGGSGGGGS (SEQ ID NO: 23) or GGGGSGGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO: 24).

[0295] In some embodiments, the adapter 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 masking domain, such as the anti-SIRPα VHH domain) includes a GS adapter located at the N-terminus and C-terminus of the protease-cleavable adapter, i.e., an adapter sequence having a sequence GS adapter-protease-cleavable adapter-GS adapter. In some embodiments, the adapter sequentially comprises the sequence GGGGS (SEQ ID NO: 11), the protease-cleavable adapter, and GGGGS (SEQ ID NO: 11) from the N-terminus to the C-terminus. In some embodiments, the adapter sequence sequentially comprises the sequence GGGGSGGGGS (SEQ ID NO: 9), the protease-cleavable adapter, and GGGGSGGGGS (SEQ ID NO: 9) from the N-terminus to the C-terminus. In some embodiments, the adapter sequence sequentially comprises the sequence GGGGS (SEQ ID NO: 11), the protease-cleavable adapter, and GGGGSGGGGS (SEQ ID NO: 9) from the N-terminus to the C-terminus. In some embodiments, the adapter sequence comprises, from N-terminus to C-terminus, the sequence: GGGGSGGGGS (SEQ ID NO: 9), a protease-cleavable adapter, and GGGGS (SEQ ID NO: 11).

[0296] In some embodiments, the total length of the linker sequence (such as a linker sequence having a sequence GS linker-protease cleavable linker-GS linker) located 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 masking domain, such as an anti-SIRPα VHH domain) does not exceed 50 amino acids. In some embodiments, the linker length is 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.

[0297] D. Target cell antigen and target cell binding domain The binders covered by this disclosure may comprise at least one binding domain for binding a target cell antigen (e.g., a cancer antigen) and at least one binding domain for binding an APP. In some embodiments, the target cell antigen is expressed on cells targeted to be acted upon by myeloid cell activity (e.g., direct and / or indirect killing). In some embodiments, the binders covered by this disclosure comprise a target cell binding domain that recognizes a target cell antigen expressed on cells targeted to regulate (e.g., direct and / or indirect killing). In some embodiments, the binders covered by this disclosure comprise two or more target cell binding domains (e.g., 2, 3, 4 or more) that recognize a target cell antigen expressed on cells targeted to regulate (e.g., direct and / or indirect killing). In some embodiments, the binders covered by this disclosure comprise two target cell binding domains, wherein each target cell binding domain recognizes a different target cell antigen expressed on cells targeted to regulate (e.g., direct and / or indirect killing). In some embodiments, the binders covered by this disclosure comprise two or more target cell binding domains (e.g., 2, 3, 4 or more) that collectively recognize at least two target cell antigens expressed on cells targeted to modulate (e.g., direct killing and / or indirect killing). 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 derived from a solid tumor.

[0298] In some implementations, 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: Table 2

[0299] *Table 2 includes: RNA nucleic acid molecules (e.g., thymine replaced by uridine); nucleic acid molecules encoding orthologs of the encoded protein; DNA or RNA nucleic acid sequences comprising nucleic acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher identity in their full length with any of the nucleic acid sequences or portions listed in Table 2; orthologs of any of the proteins listed in Table 2; and amino acid sequences comprising amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher identity in their full length with any of the amino acid sequences or portions listed in Table 2. These nucleic acid or amino acid moieties can function as full-length nucleic acid or amino acid molecules, as further described herein.

[0300] Representative antigen-binding domains are well known in the art (see, for example, U.S. Patent No. 11,459,394), and include, but are not limited to, antigen-binding domains obtained from: 3F8 targeting GD2 gangliosides, cantuzumab targeting MUC1, 8H9 targeting B7-H3, emeximab targeting GD3 gangliosides, nimotuzumab targeting EGFR; nimotuzumab targeting EGFR, cetuximab targeting EGFR, P2X targeting EGFR, 11F6 targeting EGFR, and other anti-EGFR antibodies targeting EGFR, such as erlotinib, Osimertinib, neratinib, gefitinib, panitumumab, dacomitinib, lapatinib, mobotinib, and vandetanib; fentulumab targeting IGF1R, seretumab targeting ERBB3, vanvitazumab targeting TYRP1, lmab362 targeting Cldn18.2, durituzumab targeting HER3, adenomyumab targeting EPCAM, trastuzumab targeting HER2, gelentoxici targeting CA9, nexituzumab targeting EGFR, tucotuzumab targeting EPCAM, zaltoxici targeting HER1, and 5B1 targeting Ca19-9. (See, for example, PCT Publication No. WO2015053871; Sawada et al. Clin Cancer Res. 1 March 2011; 17(5): 1024–1032), r7E3 targeting Ca19-9 (Sawada et al.) or 121SLE targeting Ca19.9 (Sawada et al.).

[0301] In the 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 a binding fragment thereof.

[0302] In some embodiments, the multispecific binding construct comprises an antibody or antigen-binding fragment selected from the following: 3F8 targeting GD2 gangliosides, cantuzumab targeting MUC1, 8H9 targeting B7-H3, emeximab targeting GD3 gangliosides, nimotuzumab targeting EGFR, nixituzumab targeting EGFR, cetuximab targeting EGFR, P2X targeting EGFR, 11F6 targeting EGFR, and IGF1 targeting... The following drugs are listed: fentolimumab (targeting R), seretumab (targeting ERBB3), vanvitumab (targeting TYRP1), lmab362 (targeting Cldn18.2), duritumab (targeting HER3), adenomyumab (targeting EPCAM), trastuzumab (targeting HER2), gilentuximab (targeting CA9), nexituzumab (targeting EGFR), tocotuzumab (targeting EPCAM), zartuximab (targeting HER), and 5B1 (targeting Ca19.9).

[0303] In some embodiments, the multispecific binding construct comprises an anti-EGFR antibody or a binding fragment thereof. In some embodiments, an 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 nexituzumab (11F8), cetuximab, nimotuzumab, and P2X. In some embodiments, the multispecific binding construct comprises an anti-EGFR antigen-binding fragment as a Fab. In some embodiments, the multispecific binding construct comprises an anti-EGFR antigen-binding fragment as a single-chain variable fragment (scFv). In some embodiments, the multispecific binding construct comprises an scFv linked to an Fc. In some embodiments, the multispecific binding construct comprises a Fab linked to an Fc. In some embodiments, the antigen-binding domain is a Fab.

[0304] In some embodiments, Fab is the Fab of nexituzumab, 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 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: 2. In some embodiments, Fab comprises: a heavy chain comprising the amino acid seq...

Claims

1. A multispecific antigen-binding construct comprising: (i) The first antigen-binding domain binds to and inhibits the 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 the APP; (iii) A connector comprising a protein-hydrolyzable cuttable connector; (iv) A third antigen-binding domain that binds to a first target cell antigen; and (v) Immunoglobulin Fc region, The connector, which includes the protein hydrolyzable cleavage connector, connects the second antigen-binding domain to the first antigen-binding domain or the immunoglobulin Fc region.

2. The multispecific antigen-binding construct of claim 1, wherein the first antigen-binding domain and the second antigen-binding domain are connected by the connector comprising the protein-hydrolyzable cleavable connector.

3. The multispecific antigen-binding construct of claim 1, wherein the immunoglobulin Fc region and the second antigen-binding domain are connected by the connector comprising the protein-cleavable cleavable connector.

4. The multispecific antigen-binding construct according to any one of claims 1-3, wherein when the adapter is in an uncleaved state, the second antigen-binding domain inhibits or reduces the binding of the first antigen-binding domain to the APP, and wherein when the adapter has been cleaved by protein hydrolysis, the second antigen-binding domain does not interfere with the binding of the first antigen-binding domain to the APP.

5. The multispecific antigen-binding construct according to any one of claims 1-4, wherein the APP is selected from the group consisting of: differentiation cluster 47 (CD47), differentiation cluster 24 (CD24), programmed cell death 1 ligand 1 (PD-L1), programmed cell death 1 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 multispecific antigen-binding construct according to any one of claims 1-5, wherein the first antigen-binding domain 1) inhibits the interaction of the APP with its binding partner on myeloid cells, or 2) inhibits the interaction of the APP with its binding partner on cells targeted to be active by myeloid cells.

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

8. The multispecific antigen-binding construct according to any one of claims 1-7, wherein the first antigen-binding domain binds to and inhibits the APP, inhibiting interactions selected from the group consisting of: the interaction between CD47 and SIRPα, the interaction between CD24 and SIGLEC10, the interaction between PD-1 and PD-1 ligands (PD-L1 or PD-L2), and the interaction between LILRB1 ligands (β-L1, β-L2 ... The interaction between β2M or the MHC-I complex and LILRB1, and the interaction between the LILRB2 ligand and LILRB2, wherein 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 a protein of the MHC-I complex (HLA-A, HLA-B, or HLA-C).

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

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

11. The multispecific antibody-binding construct of any one of claims 1-10, wherein the first antigen-binding domain comprises: (a) an extracellular domain (ECD) of a protein expressed on the cell surface; (b) a binding fragment of the ECD of the protein expressed on the cell surface; or (c) a variant of the ECD or the binding fragment of the protein expressed on the cell surface, the variant being engineered to improve binding to the APP.

12. The multispecific antibody-binding construct of claim 11, wherein the cell surface expressed protein is wild-type SIRPα, and the first antigen-binding domain comprises: (a) the ECD of the wild-type SIRPα, (b) a binding fragment of the wild-type SIRPα; or (c) a variant of the ECD of the wild-type SIRPα or the binding fragment, the variant being engineered to improve binding to the APP, wherein the APP is CD47.

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

14. The multispecific antibody-binding construct according to any one of claims 1-10, wherein the first antigen-binding domain comprises a domain of wild-type SIRPα that binds CD47 or a variant thereof, the variant comprising one or more amino acid substitutions in the domain of the wild-type SIRPα that improve binding to CD47.

15. A multispecific antigen-binding construct comprising: (i) A first antigen-binding domain comprising: (a) a domain of wild-type SIRPα that binds to an antiphagocytic protein (APP), or (b) a variant thereof, said variant comprising one or more amino acid substitutions in said domain of said wild-type SIRPα that improve binding to said APP, wherein said APP is CD47; The second antigen-binding domain, which is an anti-SIRPα antibody or an antigen-binding fragment, binds to the first antigen-binding domain and inhibits or reduces the interaction between the first antigen-binding domain and the APP. The first antigen-binding domain and the second antigen-binding domain are connected by a connector containing a protein-hydrolyzable cleavable linker.

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

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

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

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

20. The multispecific antigen-binding construct according to any one of claims 1-19, 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 1-19, 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 1-21, wherein the first antigen-binding domain is or comprises the sequence shown in SEQ ID NO:

103.

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

104.

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

27. The multispecific antigen-binding construct of claim 26, wherein the variant SIRPα binds to wild-type human CD47, whose dissociation constant (K) D Less than 100 nanomoles (nM), less than 10 nM, less than 1 nM, less than 100 picomoles (pM), less than 10 pM or less than 1 pM, or any combination of the foregoing values.

28. The multispecific antigen-binding construct of claim 26 or claim 27, wherein the variant SIRPα binds to wild-type human CD47, whose dissociation constant (K) D Less than 100 nanomoles (nM), optionally 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.

29. The multispecific antigen-binding construct of claim 26 or claim 27, wherein the variant SIRPα binds to wild-type human CD47, whose dissociation constant (K0) D Below 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.

30. The multispecific antigen-binding construct of claim 26 or claim 27, wherein the variant SIRPα binds to wild-type human CD47, whose dissociation constant (Ki) D () Less than 100 picomoles (pM).

31. The multispecific antigen-binding construct of claim 26, claim 27 or claim 30, wherein the variant SIRPα binds to wild-type human CD47, and its dissociation constant (K) D The range is 1pM to 100pM, optionally 1pM to 75pM, 1pM to 50pM, 1pM to 25pM, 1pM to 10pM, 10pM to 100pM, 10pM to 75pM, 10pM to 50pM, 10pM to 25pM, 25pM to 100pM, 25pM to 75pM, 25pM to 50pM, or 50pM to 100pM, 50pM to 75pM, or 75pM to 100pM.

32. The multispecific antigen-binding construct according to any one of claims 14, 15, and 26-31, wherein the 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 SEQ ID NO: Amino acid number 103 or SEQ ID NO:

104.

33. The 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 number 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 comprise K53R, E54Q and S66T (L66T), corresponding to the amino acid numbers of SEQ ID NO: 103 or SEQ ID NO:

104.

35. The multispecific antigen-binding construct according to any one of claims 14, 15, and 26-34, wherein 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, 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 amino acid numbers in 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 number of SEQ ID NO: 103 or SEQ ID NO:

104.

37. The multispecific antigen-binding construct according to any one of claims 1-21, 23, 24 and 26-36, 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 1-21, 23, 24 and 26-36, 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 1-21, 23, 24 and 26-38, wherein the first antigen-binding domain is or comprises the amino acid sequence shown in SEQ ID NO:

105.

40. The multispecific antigen-binding construct according to any one of claims 1-39, 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 number 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 or comprises the amino acid sequence shown in SEQ ID NO:

10.

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 or comprises the amino acid sequence shown in SEQ ID NO:

27.

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

49. The multispecific antigen-binding construct of claim 48, wherein 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.

50. The multispecific antigen-binding construct of claim 48 or claim 49, wherein the first antigen-binding domain is a single-domain antibody serving as VHH.

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

52. The multispecific antigen-binding construct according to any one of claims 1-10 and 48-51, 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 1-10 and 48-52, wherein the first antigen binding comprises the sequence shown in SEQ ID NO:

208.

54. The multispecific antigen-binding construct according to any one of claims 1-53, wherein the dissociation constant of the second antigen-binding domain binding to the first antigen-binding domain is higher than the dissociation constant of the first antigen-binding domain binding to the APP.

55. The multispecific antigen-binding construct of claim 54, wherein the dissociation constant (Kd) of the second antigen-binding domain 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 that of the dissociation constant of the first antigen-binding domain to the APP.

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

57. The multispecific antigen-binding construct according to any one of claims 1-56, wherein the dissociation constant of the second antigen-binding domain binding to the first antigen-binding domain is 1 nM or higher, optionally 100 nM to 1 µM, 10 nM to 1 µM or 1 nM to 1 µM.

58. The multispecific antigen-binding construct according to any one of claims 1-57, wherein the dissociation constant of the second antigen-binding domain binding to the first antigen-binding domain is 1 nM or higher.

59. The multispecific antigen-binding construct according to any one of claims 1-58, wherein the dissociation constant of the second antigen-binding domain binding to the first antigen-binding domain is 10 nM or higher.

60. The multispecific antigen-binding construct according to any one of claims 1-58, wherein the dissociation constant of the second antigen-binding domain binding to the first antigen-binding domain is 100 nM or higher.

61. The multispecific antigen-binding construct according to any one of claims 1-58, wherein the dissociation constant of the second antigen-binding domain binding to the first antigen-binding domain is 1 µM or higher.

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

64. The multispecific antigen-binding construct of claim 15, claim 62 or claim 63, wherein 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, sdFv.

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

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

67. The multispecific antigen-binding construct of claim 65 or claim 66, wherein the VHH comprises: a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44 and 45; a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from SEQ ID NO: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60; and a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73.

68. The multispecific antigen-binding construct according to any one of claims 65-67, wherein the SIRPα VHH domain comprises CDR1, CDR2, and CDR3 as follows: SEQ ID NO: 37, 46, and 61, respectively; SEQ ID NO: 38, 46, and 61, respectively; SEQ ID NO: 39, 47, and 62, respectively; SEQ ID NO: 40, 48, and 63, respectively; SEQ ID NO: 41, 49, and 64, respectively; SEQ ID NO: 37, 50, and 61, respectively; SEQ ID NO: 42, 51, and 65, respectively; SEQ ID NO: 43, 52, and 66, respectively; SEQ ID NO: 37, 53, and 67, respectively; SEQ ID NO: 44, 54, and 68, respectively; SEQ ID NO: 43, 55, and 63, respectively; SEQ ID NO: 40, 56, and 69, respectively; SEQ ID NO: 37, 57, and 70, respectively; SEQ ID NO: 40, 55 and 63; SEQ ID NO: 41, 58 and 71 respectively; SEQ ID NO: 43, 59 and 72 respectively; SEQ ID NO: 37, 60 and 73 respectively; or SEQ ID NO: 45, 56 and 73 respectively.

69. The multispecific antigen-binding construct according to any one of claims 65-68, wherein the VHH domain comprises the amino acid sequence shown in any one of SEQ ID NO: 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 NO: 13-21 and 28-36, and binds SIRPα.

70. The multispecific antigen-binding construct according to any one of claims 65-68, wherein the VHH domain comprises the amino acid sequence shown in any one of SEQ ID NO: 13-21 and 28-36.

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

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

73. The multispecific antigen-binding construct of claim 71 or claim 72, wherein the at least one variant SIRPα comprises one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improve binding to CD47.

74. The multispecific antigen-binding construct according to any one of claims 71-73, wherein the IgV domain of the wild-type human SIRPα or a variant thereof is selected from: (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 SEQ ID NO: 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 SEQ ID NO: 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) 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.

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

76. The multispecific antigen-binding construct according to any one of claims 71-75, wherein the anti-SIRPα antibody or antigen-binding fragment binds to wild-type human SIRPα, optionally the IgV domain of the wild-type human SIRPα, wherein the wild-type human SIRPα comprises: (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, optionally comprising wild-type human SIRPα of 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, optionally comprising SEQ ID NO: The amino acid sequence shown in 104 represents wild-type human SIRPα.

77. The multispecific antigen-binding construct according to any one of claims 71-75, wherein the anti-SIRPα antibody or antigen-binding fragment binds to the variant SIRPα, optionally the IgV domain of the variant SIRPα, wherein the variant SIRPα comprises one or more amino acid substitutions selected from the group consisting of wild-type 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 amino acid numbers of SEQ ID NO: 103 or SEQ ID NO:

104.

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

104.

79. The multispecific antigen-binding construct of claim 77 or claim 78, wherein 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, 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 amino acid numbers in SEQ ID NO: 103 or SEQ ID NO:

104.

80. The multispecific antigen-binding construct according to any one of claims 77-79, 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 numbers of SEQ ID NO: 103 or SEQ ID NO:

104.

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

82. The multispecific antigen-binding construct according to any one of claims 71-81, wherein the anti-SIRPα antibody or antigen-binding fragment binds to the variant SIRPα, optionally the IgV domain of the variant SIRPα, the variant SIRPα comprising: (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: 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; or (v) 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.

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

84. The multispecific antigen-binding construct according to any one of claims 1-83, wherein the linker is a substrate of a protease, optionally wherein the protease is an extracellular protease, and / or the linker is a substrate of renin, pepsin C, aspartate protease A, matrix metalloproteinase (MMP), matrix protease, urokinase-type plasminogen activator (uPA), integrin metalloproteinase (ADAM), integrin metalloproteinase containing a platelet-reactive protein motif (ADAMTS), podocyte oleoresin, urokinase, or hepatic serine.

85. The multispecific antigen-binding construct according to any one of claims 1-84, wherein the proteolytically cleavable linker is a polypeptide that functions as a substrate of a protease.

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

87. The multispecific antigen-binding construct of claim 85 or claim 86, wherein the protease is selected from matrix proteases, matrix metalloproteinases (MMPs), granzyme B, and combinations thereof.

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

89. The multispecific antigen-binding construct according to any one of claims 1-88, wherein the protein-hydrolyzable cleavable linker is VHMPLGFLGPRQARVVN (SEQ ID NO: 22).

90. The multispecific antigen-binding construct of any one of claims 1-89, wherein the adapter comprising the protein hydrolyzable cleavable adapter comprises an N-terminal and / or C-terminal GS adapter sequence.

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

92. The multispecific antigen-binding construct according to any one of claims 15-47 and 54-91, 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 1-14 and 16-92, wherein the first target cell antigen is expressed on cells targeted to be acted upon by myeloid cell activity.

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

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

96. The multispecific antigen-binding construct according to any one of claims 1-14 and 16-95, 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 1-14 and 16-96, 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 1-14 and 16-97, wherein the third antigen-binding domain is Fab.

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

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

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

102. The multispecific antigen-binding construct according to any one of claims 1-14, 16-97, 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 99-102, wherein the third antigen-binding domain comprises two different single-chain antibody fragments.

104. The multispecific antigen-binding construct of claim 103, wherein the third antigen-binding domain is a double complementary site.

105. The multispecific antigen-binding construct according to any one of claims 1-104, further comprising a fourth antigen-binding domain for binding a second target cell antigen, optionally wherein the second target cell antigen is expressed on the cells targeted to be acted upon by myeloid cell activity.

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

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

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

109. The multispecific antigen-binding construct according to any one of claims 103-108, 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 103-109, 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. The multispecific antigen-binding construct according to any one of claims 15-110, further comprising an immunoglobulin Fc region.

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

113. The multispecific antigen-binding construct of claim 112, wherein the third antigen-binding domain is bivalent.

114. The multispecific antigen-binding construct of claim 112 or claim 113, wherein the first antigen-binding domain is bivalent.

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

116. The multispecific antigen-binding construct according to any one of claims 1-14 and 16-115, 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 1-14 and 16-116, wherein the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO:

98.

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

119. The multispecific antigen-binding construct of 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 multispecific antigen-binding construct of claim 118 or claim 119, wherein the variant Fc region comprises one or more amino acid mutations at positions selected from the group consisting of: 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, 2 93, 295, 296, 297, 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, optionally one or more of the one or more amino acid mutations are one or more amino acid substitutions, and optionally the one or more amino acid substitutions are selected from the group consisting of: 220S, 229S, 232G, 233P, 234A, 234D, 234E, 234F, 234G, 234H, 234L, 234N, 234Q, 234T, 234V, 234Y, 235A , 235D, 235E, 235F, 235G, 235H, 235N, 235P, 235Q, 235R, 235S, 235T, 235W , 235Y, 236A, 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 , 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, 305I, 309L, 316D, 318A, 324T, 325A, 325E, 325H, 325L, 325Q, 325T, 325V, 326W, 327G, 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, 331D, 331E, 331F, 331G, 331H, 331K, 331L, 331M, 331N, 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, 121. The multispecific antigen-binding construct of 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 of 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 of claim 121 or claim 122, wherein the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO:

97.

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

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

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

127. The multispecific antigen-binding construct according to any one of claims 1-14 and 16-126, wherein the third antigen-binding region is Fab, and the multispecific antigen-binding construct comprises: A first polypeptide, comprising, from its N-terminus to its C-terminus, the following: a heavy chain variable region (VH) and a heavy chain constant region (CH1) of the 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 the proteolytically cleavable linker shown in SEQ ID NO: 12; and a second antigen-binding domain comprising a sequence having at least 95% sequence identity with the sequences shown in any one of SEQ ID NO: 13-21 and 28-36; and The second polypeptide comprises the light chain variable region (VL) and light chain constant region (CL) of the Fab.

128. The multispecific antigen-binding construct according to any one of claims 1-14 and 16-126, wherein the third antigen-binding region is Fab, and the multispecific antigen-binding construct comprises: A first polypeptide, comprising, from its N-terminus to its C-terminus, the following: a heavy chain variable region (VH) and a heavy chain constant region (CH1) of the 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 the proteolytically cleavable linker shown in SEQ ID NO: 12; and a second antigen-binding domain comprising a sequence having at least 95% sequence identity with the sequences shown in any one of SEQ ID NO: 13-21 and 28-36; and The second polypeptide comprises the light chain variable region (VL) and light chain constant region (CL) of the Fab.

129. The multispecific antigen-binding construct of claim 128, wherein the second antigen-binding domain comprises the sequence shown in any one of SEQ ID NO: 13-21 and 28-36.

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

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

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

133. The multispecific antigen-binding construct of claim 131, wherein the GS adapter is GGGGS (SEQ ID NO: 11), GGGGSGGGGS (SEQ ID NO: 9), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 23), or GGGGSGGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO: 24).

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

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

136. The multispecific antigen-binding construct according to any one of claims 127-135, wherein the Fab comprises: (a) A heavy chain comprising 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 comprising 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 comprising 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 amino acids 1-217 of SEQ ID NO: 94; (d) A heavy chain comprising 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 comprising 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.

137. The multispecific antigen-binding construct according to any one of claims 127-136, wherein the Fab comprises: (a) 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; (b) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 205, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 2; (c) A heavy chain comprising amino acids 1-217 of SEQ ID NO: 93, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 94; (d) A heavy chain comprising amino acids 1-221 of SEQ ID NO: 211, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 96; or (e) A heavy chain comprising amino acids 1-217 of SEQ ID NO: 212, and a light chain comprising the amino acid sequence shown in SEQ ID NO:

213.

138. The multispecific antigen-binding construct of any one of claims 127-137, wherein the Fab is a nexituzumab Fab, comprising: a heavy chain comprising a 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.

139. The multispecific antigen-binding construct according to any one of claims 127-138, wherein the Fab is a nexituzumab Fab comprising: 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.

140. The multispecific antigen-binding construct of any one of claims 127-137, wherein the Fab is a nexituzumab Fab, comprising: a heavy chain comprising a 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.

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

2.

142. The multispecific antigen-binding construct according to any one of claims 1-14, 16-21, 23, 24, 40-44, 48-127, and 129-141, wherein the multispecific polypeptide construct comprises: a first polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in any one of SEQ ID NO: 110-120, and a second polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:

2.

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

2.

144. The multispecific antigen-binding construct according to any one of claims 1-14, 16, 20-21, 23, 24, 26-38, 40-43, 45-127 and 129-141, wherein the multispecific polypeptide construct comprises: a first polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in any one of SEQ ID NO: 125-137, and a second polypeptide chain comprising a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:

2.

145. The multispecific antigen-binding construct according to any one of claims 1-14, 16, 20-21, 23, 24, 26-38, 40-43, 45-127, 129-141, and 144, wherein the multispecific polypeptide construct comprises: a first polypeptide chain comprising the sequence shown in any one of SEQ ID NO: 125-137, and a second polypeptide chain comprising the sequence shown in SEQ ID NO:

2.

146. The multispecific antigen-binding construct according to any one of claims 1-14 and 16-97, 99-104, 111-123, wherein 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, the Fc region, the first antigen-binding domain, the adapter comprising the protein-cleavable adapter, and the second antigen-binding domain.

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

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

149. The multispecific antigen-binding construct of 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 multispecific antigen-binding construct of claim 148 or claim 148, wherein the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO:

102.

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

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

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

154. The multispecific antigen-binding construct of claim 153, wherein the scFv comprises: a VH chain comprising an amino acid sequence having at least 95% sequence identity with the variable heavy (VH) chain sequence presented in SEQ ID NO: 207, and a VL chain comprising an amino acid sequence having at least 95% sequence identity with the variable light (VL) chain sequence presented in SEQ ID NO:

207.

155. The multispecific antigen-binding construct of claim 153 or the multispecific antigen-binding construct of 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 153-155, 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 1-122, wherein the Fc region is a heterodimeric Fc region.

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

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

160. The multispecific antigen-binding construct of claim 158, wherein the first antigen-binding domain is monovalent and the third antigen-binding domain is divalent.

161. The multispecific antigen-binding construct according to any one of claims 1-122 and 157-160, comprising: a first antigen-binding domain binding to an antiphagocytic protein (APP), a second antigen-binding domain binding 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 serving as a target cell antigen-binding domain, binding to a target cell antigen expressed on a cell targeted for myeloid cell activity, wherein the second antigen-binding domain is linked to one of the first Fc polypeptide or the second Fc polypeptide via a proteolytically cleavable linker.

162. The multispecific antigen-binding construct of claim 161, comprising: (1) A first heavy chain comprising a first polypeptide chain of the heterodimer Fc and the first antigen-binding domain; and (2) A second heavy chain comprising the second polypeptide chain of the heterodimer Fc, a linker comprising the protein-cleavable linker, and the second antigen-binding domain. The first heavy chain and the second heavy chain, or both of them, contain the third antigen-binding domain or a chain thereof.

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

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

165. The multispecific antigen-binding construct of any one of claims 157-164, wherein the third antigen-binding domain is Fab, and the multispecific antigen-binding construct comprises: The first polypeptide comprises, from the N-terminus to the C-terminus, the heavy chain variable region (VH) and heavy chain constant region 1 (CH1) of the Fab, the first polypeptide of the Fc region of the heterodimeric immunoglobulin, and the first antigen-binding domain. The second polypeptide, comprising, from its N-terminus to its C-terminus, the VH and CH1 of the Fab, the second polypeptide of the heterodimer Fc region, the linker comprising the protein-hydrolyzable cleavable linker, and the second antigen-binding domain; and The third polypeptide comprises the light chain variable region (VL) and light chain constant region (CL) of the Fab.

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

167. The multispecific antigen-binding construct of claim 166, wherein one or more amino acid substitutions are mortar-and-mortar modifications or charge mutations to enhance the electrostatic complementarity of the polypeptide.

168. The multispecific antigen-binding construct of claim 166 or claim 167, wherein the first Fc polypeptide in the heterodimeric Fc region comprises an amino acid substitution selected from Thr366Ser, Leu368Ala, Tyr407Val and combinations thereof, and the second Fc polypeptide in the heterodimeric Fc region comprises the amino acid substitution T366W, and optionally wherein the first Fc polypeptide and the second Fc polypeptide further comprise an amino acid substitution in which a non-cysteine ​​residue is replaced by a cysteine ​​residue, wherein the amino acid substitution of the first polypeptide is located at one of positions Ser354 and Y349, and the amino acid substitution of the second Fc polypeptide is located at the other of positions Ser354 and Y349.

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

170. The multispecific antigen-binding construct according to any one of claims 166-169, 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 166-169, 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. The multispecific antigen-binding construct according to any one of claims 157-171, comprising: The first polypeptide comprises, from N-terminus to C-terminus, the heavy chain variable region (VH) and heavy chain constant region (CH1) of the Fab, the first immunoglobulin Fc region comprising the amino acid sequence shown in SEQ ID NO: 106, and the first antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO:

27. The second polypeptide, comprising, from its N-terminus to its C-terminus, the VH and CH1 of the Fab, a second immunoglobulin Fc region comprising the amino acid sequence shown in SEQ ID NO: 107, a linker comprising the proteolytically cleavable linker shown in SEQ ID NO: 12, and a second antigen-binding domain comprising a sequence having at least 95% sequence identity with the sequences shown in any of SEQ ID NO: 13-21 and 28-36; and The third polypeptide comprises the light chain variable region (VL) and light chain constant region (CL) of the Fab.

173. The multispecific antigen-binding construct of claim 172, wherein the second antigen-binding domain comprises the sequence shown in any one of SEQ ID NO: 13-21 and 28-36.

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

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

176. The multispecific antigen-binding construct of claim 174 or claim 175, wherein the Fab comprises: (a) A heavy chain comprising 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 comprising 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 comprising 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 amino acids 1-217 of SEQ ID NO: 94; (d) A heavy chain comprising 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 comprising 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.

177. The multispecific antigen-binding construct according to any one of claims 174-176, wherein the Fab comprises: (a) 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; (b) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 205, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 2; (c) A heavy chain comprising amino acids 1-217 of SEQ ID NO: 93, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 94; (d) A heavy chain comprising amino acids 1-221 of SEQ ID NO: 211, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 96; or (e) A heavy chain comprising amino acids 1-217 of SEQ ID NO: 212, and a light chain comprising the amino acid sequence shown in SEQ ID NO:

213.

178. The multispecific antigen-binding construct of any one of claims 174-177, wherein the Fab is a nexituzumab Fab, comprising: a heavy chain comprising a 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.

179. The multispecific antigen-binding construct according to any one of claims 174-178, wherein the Fab is a nexituzumab Fab comprising: 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.

180. The multispecific antigen-binding construct of any one of claims 174-177, wherein the Fab is a nexituzumab Fab, comprising: a heavy chain comprising a 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.

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

2.

182. A nucleic acid encoding a multispecific antigen-binding construct as described in any one of claims 1-181.

183. The nucleic acid of claim 182, wherein it is a polycistronic sequence, wherein the nucleic acid encoding each polypeptide of the construct is separated by polycistronic elements.

184. The nucleic acid of claim 183, wherein the polycistronic element is a 2A cleavage sequence or an IRES element, optionally wherein the 2A cleavage sequence is a P2A or T2A sequence.

185. An expression vector comprising the nucleic acid as described in any one of claims 182-184.

186. A cell comprising the expression vector as described in claim 185.

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

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

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

190. A method for regulating the myeloid cell activity of myeloid cells against target cells, the method comprising: In the presence of myeloid cells, a target cell population is contacted with a multispecific antigen-binding construct as described in any one of claims 1-181, wherein the amount of the multispecific antigen-binding construct is sufficient to modulate the myeloid cell activity of the myeloid cells in relation to the target cells.

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

192. The method of claim 190 or 191, wherein the target cell is infected with a microorganism or expresses a microbial antigen.

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

194. The method of claim 190 or 191, wherein the cell is a cancer cell, or the cell expresses a tumor-associated antigen (TAA).

195. The method of 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 suffering from a microbial infection, the method comprising administering to the subject an effective amount of a multispecific antigen-binding construct as described in any one of claims 1-181 or a pharmaceutical composition as described in claim 189, thereby treating the subject for the microbial infection.

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

198. The method of claim 196 or claim 197, wherein the cancer is adenocarcinoma, bile duct (cholecystectomy) carcinoma, bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid, 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.

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

200. The method of 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 or inflammatory disease in a subject, the method comprising administering to the subject an effective amount of a multispecific antigen-binding construct as described in any one of claims 1-181 or a pharmaceutical composition as described in claim 189, thereby treating the subject's autoimmune or inflammatory disease and / or delaying its progression.

202. The method of claim 201, wherein the autoimmune or inflammatory disease is atherosclerosis, obesity, inflammatory bowel disease (IBD), Lyme disease, Hashimoto's thyroiditis, autoimmune uveitis, autoimmune valvular heart 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, alopecia areata, vasculitis and temporal arteritis, graft-versus-host disease. (GVHD), asthma, COPD, eosinophilia, conjunctivitis, glomerulonephritis, autoimmune nephritis, paraneoplastic autoimmune disease, cartilage inflammation, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, generalized juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronephropathy, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile Psoriatic arthritis, fibrotic diseases, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, generalized flare-ups of rheumatoid arthritis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, psoriasis, plaque psoriasis, guttate psoriasis, reverse psoriasis 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 abortion.

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

204. The method of 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, pulmonary heart disease, 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 disease in a subject, the method comprising administering to the subject an effective amount of a multispecific antigen-binding construct as described in any one of claims 1-181 or a pharmaceutical composition as described in claim 189, thereby treating the subject's neurological disease and / or delaying its progression.

206. The method of claim 205, wherein the neurological disease is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, an 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 of any one of claims 196-206, wherein the subject is a mammal.

208. The method of claim 207, wherein the mammal is a human, a non-human primate, a farm animal, a domesticated animal, or a laboratory animal.

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

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

211. A SIRPα-binding molecule comprising at least one heavy-chain-only variable domain (SIRPα VHH domain), said domain comprising: a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, and 45; a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from SEQ ID NO: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.

212. The SIRPα binding molecule of claim 211, wherein the at least one SIRPα VHH domain comprises CDR1, CDR2, and CDR3 as shown below: SEQ ID NO: 37, 46, and 61 respectively; SEQ ID NO: 38, 46, and 61 respectively; SEQ ID NO: 39, 47, and 62 respectively; SEQ ID NO: 40, 48, and 63 respectively; SEQ ID NO: 41, 49, and 64 respectively; SEQ ID NO: 37, 50, and 61 respectively; SEQ ID NO: 42, 51, and 65 respectively; SEQ ID NO: 43, 52, and 66 respectively; SEQ ID NO: 37, 53, and 67 respectively; SEQ ID NO: 44, 54, and 68 respectively; SEQ ID NO: 43, 55, and 63 respectively; SEQ ID NO: 40, 56, and 69 respectively; SEQ ID NO: 37, 57, and 70 respectively; SEQ ID NO: 40, 55 and 63; SEQ ID NO: 41, 58 and 71 respectively; SEQ ID NO: 43, 59 and 72 respectively; SEQ ID NO: 37, 60 and 73 respectively; or SEQ ID NO: 45, 56 and 73 respectively.

213. The SIRPα binding molecule of claim 211 or claim 212, wherein the SIRPα is human SIRPα.

214. The SIRPα-binding molecule according to any one of claims 211-213, wherein the at least one SIRPαVHH domain comprises an amino acid sequence shown in any one of SEQ ID NO: 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 NO: 13-21 and 28-36, and binds SIRPα.

215. The SIRPα binding molecule according to any one of claims 211-214, wherein the at least one SIRPαVHH domain comprises the amino acid sequence shown in any one of SEQ ID NO: 13-21 and 28-36.

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

217. The SIRPα binding molecule according to any one of claims 211-216, 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 of any one of claims 211-217, wherein the VHH domain binds to one or more IgV domains of wild-type human SIRPα or variants thereof.

219. The SIRPα binding molecule according to any one of claims 211-218, wherein the IgV domain of one or more wild-type human SIRPα or variants thereof is selected from: (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 SEQ ID NO: 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 SEQ ID NO: 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) 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.

220. The SIRPα binding molecule of any one of claims 211-219, wherein the VHH is panreactive and binds wild-type SIRPα and at least one variant SIRPα, the variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα to improve binding to CD47.

221. The SIRPα binding molecule of claim 220, wherein the VHH binds (1) the IgV domain of wild-type allele SIRPα, optionally wild-type allele 1 and / or wild-type allele 2 SIRPα, and (2) at least one IgV domain of variant SIRPα, said variant SIRPα (a) comprising one or more amino acid substitutions in the IgV domain of said wild-type SIRPα that improve binding to CD47; and / or (b) is a deglycosylated variant.

222. The SIRPα binding molecule of claim 221, wherein the IgV domain of the wild-type human SIRPα, optionally the wild-type human SIRPα, comprises: (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 SIRPα binding molecule of claim 221, wherein the variant SIRPα, optionally, the IgV domain of the variant SIRPα comprises one or more amino acid substitutions 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 amino acid numbers of SEQ ID NO: 103 or SEQ ID NO:

104.

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

104.

225. The SIRPα-binding molecule of claim 223 or claim 224, wherein 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, 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 amino acid numbers in SEQ ID NO: 103 or SEQ ID NO:

104.

226. The SIRPα-binding molecule according to any one of claims 223-225, 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 number of SEQ ID NO: 103 or SEQ ID NO:

104.

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

228. The SIRPα binding molecule as claimed in any one of claims 222-227, wherein the variant SIRPα, optionally, the IgV domain of said variant 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: 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 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.

229. A binding molecule comprising the SIRPα binding molecule as described in any one of claims 211-228 and a second binding domain for binding a second antigen.

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

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

232. The binding molecule of 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 of any one of claims 229-231, wherein the binding domain binding the second antigen is an antibody or an antigen-binding fragment.

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

235. A nucleic acid encoding a SIRPα-binding molecule as described in any one of claims 211-228.

236. A nucleic acid encoding a binding molecule as described in any one of claims 229-234.

237. An expression vector comprising the nucleic acid as described in claim 235 or claim 236.

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

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

240. The method of claim 239, further comprising isolating the SIRPα-binding molecule from the cells or population of cells, or from the culture medium in which the cells or population of cells are cultured.

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

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

243. The method of claim 242, wherein the cancer is adenocarcinoma, bile duct (biliary tract) carcinoma, bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid, 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 of claim 242 or claim 243, further comprising administering to the subject an additional therapeutic agent for treating the cancer.

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

Images