Therapeutic binding agents that conditionally promote myeloid cell activity against target cells and uses thereof

Abstract

The present disclosure provides, among other things, therapeutic binding agents that can direct activity of myeloid cells to a target cell population, such as cancer cells.

Description

THERAPEUTIC BINDING AGENTS THAT CONDITIONALLY PROMOTE MYELOID CELL ACTIVITY AGAINST TARGET CELLS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional application No.63 / 532,538 filed August 14, 2023, the contents of which are incorporated by reference in their entireties. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 336402000140SeqList.XML, created August 12, 2024, which is 461,658 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. BACKGROUND

[0003] Myeloid cells are immune cells found in virtually all tissues, contributing to immune responses and cellular maintenance. In tumor environments, myeloid cells are the most abundant immune cell type and exert 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 antitumor responses, e.g., by phagocytosis of cancer cells. SUMMARY

[0004] The present disclosure provides, among other things, therapeutic binding agents that can direct the activity of myeloid cells to a target cell population, such as direct killing of unwanted cells such as cancer cells or target immune cells, indirect killing of unwanted cells such as cancer cells or target immune cells, inhibition of target cells such as target immune cells, and the like. The present disclosure provides, among other things, therapeutic binding agents that can cause direct or indirect elimination of target cells, e.g., by directing the activity of myeloid cells (e.g., phagocytic activity of myeloid cells) to a target cell population, by antibody-dependent cellular cytotoxicity (ADCC), and / or by activities of the therapeutic binding agent that directly kill target cells. In various embodiments, target cells can be, e.g., cancer cells or target immune cells. The present disclosure further provides that, in some embodiments, such therapeutic binding agents selectively direct myeloid cells to target a cell population, such as cancer cells, in a particular context, such as in a tumor environment. In some embodiments, a therapeutic binding agent encompassed by the present disclosure directs elimination of target cells, e.g., by directing activity of myeloid cells (e.g., phagocytic activity) to a target cell population in a particular environment (e.g., to cancer cells in a tumor environment, autoimmune cells in an inflammatory environment, etc.) through a combination of elements that can include: (1) at leastone binding domain that binds an anti-phagocytic protein (APP), (e.g., as expressed on myeloid cells or cells targeted for killing or inhibition of an unwanted activity) (APP-binding domain), (2) at least one binding domain that conditionally inhibits interaction of the anti-APP binding domain with its target (mask domain), and (3) a proteolytically cleavable linker positioned such that its cleavage relieves the inhibition of APP binding by the mask domain Therapeutic binding agents encompassed by the present disclosure can comprise one or more additional elements, such as, for example, 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 a protease characteristic of and / or specific to the particular environment of interest (e.g., a tumor microenvironment), thereby achieving activatable activity. The present disclosure includes the discovery that a therapeutic binding agent according to the present disclosure can selectively direct elimination of target cells, e.g., by directing myeloid cells in tumor environments to target and / or phagocytose target cells, by antibody-dependent cellular cytotoxicity (ADCC), and / or by activities of the therapeutic binding agent that directly kill target cells. In various embodiments, the target cells are cancer cells and therapeutic binding agents provided herein are useful in the treatment of cancer. In various embodiments, therapeutic binding agents provided herein modulate target cell activity in a particular environment of interest (e.g., inhibit an unwanted activity such as inhibit inflammatory and / or autoimmune function of immune cell subtypes and / or of antigen- specific immune cells).

[0005] Methods and compositions encompassed by the present disclosure are associated with numerous advantages. For example, the present disclosure appreciates that APP blockade can direct activity of myeloid cells (e.g., phagocytic activity) to target cells of interest, such as cancer cells, but that such blockade can cause myeloid cells in healthy tissue to target and destroy healthy cells, with deleterious and / or toxic effects. This challenge is particularly acute because many APPs are present (e.g., ubiquitously present) on both normal and target cells of interest, such as cancer cells. Various therapeutic binding agents encompassed by the present disclosure include a mask domain that is associated with a proteolytically cleavable linker and is positioned such that it suppresses APP binding by an APP-binding domain until such suppression is relieved by cleavage of the linker. Accordingly, various therapeutic binding agents encompassed by the present disclosure advantageously provide selective targeting of APP-expressing (e.g., myeloid) cells in a target environment of interest (e.g., a 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 spares healthy cells and improves pharmacokinetic properties (e.g., therapeutic index), safety, and / or efficacy against target cells of interest (e.g., cancer cells).

[0006] In some embodiments, a therapeutic binding agent of the present disclosure causes elimination of target cells by antibody-dependent cellular phagocytosis (ADCP). In some embodiments, a therapeutic binding agent of the present disclosure causes elimination of target cellsby antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, a therapeutic binding agent of the present disclosure causes elimination of target cells by direct killing (also referred to herein as direct elimination). In some embodiments, therapeutic binding agents encompassed by the present disclosure direct or promote an activity of myeloid cells (e.g., conditionally direct or promote an activity of myeloid cells to a target cell population) selected from the group consisting of phagocytosis (e.g., antibody-dependent cellular phagocytosis (ADCP)), direct cell-mediated killing activity (e.g., antibody-dependent cellular cytotoxicity (ADCC), promoting cytotoxic activity by lymphocytes, etc.), and indirect cell killing activity (e.g., promoting complement-dependent cytotoxicity (CDC), reducing immune checkpoint repression of lymphocytes, reducing immune checkpoint repression of myeloid cells, etc.). In some embodiments, therapeutic binding agents encompassed by the present disclosure direct or promote a particular activity of myeloid cells (e.g., conditionally direct or promote activity of myeloid cells to a target cell population) such as 1) phagocytosis, but not direct cell-mediated killing activity or indirect cell killing activity, 2) direct cell- mediated killing activity but not phagocytosis or indirect cell killing activity, and 3) indirect cell killing activity, but not phagocytosis or direct cell-mediated killing activity. In some embodiments, therapeutic binding agents encompassed by the present disclosure modulate an activity of myeloid cells (e.g., conditionally directed activity of myeloid cells on a target cell population) on a target cell involving inhibiting an unwanted activity, such as inhibiting pro-inflammatory and / or autoimmune function of immune cell subtypes and / or of antigen-specific immune cells. In some embodiments, therapeutic binding agents encompassed by the present disclosure modulate an activity of myeloid cells (e.g., conditionally directed activity of myeloid cells on a target cell population) on a target cell involving promoting a desired activity, such as promoting anti-inflammatory and / or anti-autoimmune function of immune cell subtypes and / or of antigen-specific immune cells.

[0007] In at least certain aspects, the present disclosure provides a multispecific antigen binding construct including: (i) a first antigen binding domain that binds to and inhibits an anti- phagocytic 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 the APP; where the first antigen binding domain and the second antigen binding domain are joined by a proteolytically cleavable linker.

[0008] In at least certain aspects, the present disclosure provides a multispecific antigen binding construct including: (i) a first antigen binding domain that binds to and inhibits an anti- phagocytic protein (APP); and (ii) a second antigen binding domain that binds to the first antigen binding domain; where the first antigen binding domain and the second antigen binding domain are joined by a proteolytically cleavable linker, where when the linker is in an uncleaved state, the second antigen-binding domain inhibits or reduces the binding of the first antigen-binding domain to the APP, and where 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 the APP.

[0009] In at least certain aspects, the present disclosure provides a multispecific antigen binding construct comprising: (i) a first antigen binding domain that binds to and inhibits an anti- phagocytic 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 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, wherein the linker comprising the proteolytically cleavable linker joins the second antigen binding domain to the first antigen binding domain or to the immunoglobulin Fc region.

[0010] In various embodiments, the first antigen binding domain and the second antigen binding domain are joined by the linker comprising the proteolytically cleavable linker. In various embodiments, the immunoglobulin Fc region and the second antigen binding domain are joined by the linker comprising the proteolytically cleavable linker. In various embodiments, where the linker is in an uncleaved state, the second antigen-binding domain inhibits or reduces the binding of the first antigen-binding domain to the APP, and where 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 the APP.

[0011] In various embodiments, the APP is selected from the group including or consisting of: cluster of differentiation 47 (CD47), cluster of differentiation 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 a binding partner thereof on a myeloid cell or 2) inhibits the interaction of the APP with a binding partner thereof on a cell targeted for modulation (e.g., killing, e.g., by phagocytosis). In various embodiments, the myeloid cell is a macrophage, a dendritic cell, a monocyte, a neutrophil, a tumor associated macrophage (TAM), a tumor infiltrating macrophage (TIM), or a myeloid-derived suppressor cell (MDSC). In various embodiments, the first antigen binding domain inhibits an interaction selected from the group including or consisting of the interaction between CD47 and SIRPα, the interaction between CD24 and SIGLEC10, the interaction between PD-1 and a PD-1 ligand (PD-L1 or PD-L2), the interaction between an LILRB1 ligand (β 2M or MHC-I complex) and LILRB1, and the interaction between an LILRB2 ligand and LILRB2, optionally where 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 domainor 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 an MHC-I complex (HLA-A, HLA-B, or HLA-C).

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

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

[0014] In various embodiments, the first antigen binding domain is 100 to 120 amino acids in length, optionally 106 to 118 amino acids in length, more optionally 112 to 118 amino acids in length. 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, the wild-type SIRPα is a wild-type human SIRPα. In various embodiments, the first antigen binding domain comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:103. In various embodiments, the first antigen binding domain is set forth by an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to SEQ ID NO:103. In various embodiments, the first antigen binding domain is or comprises the sequence set forth in SEQ ID NO: 103.

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

[0016] In various embodiments, the first antigen binding domain is a variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47. In various embodiments, the variant SIRPα binds to a wildtype human CD47 with a dissociation constant (KD) of less than 100 nanomolar (nM), less than 10 nM, less than 1 nM, less than 100 picomolar (pM), less than 10 pM or less than 1 pM, or any combination between any of the foregoing. In various embodiments, the variant SIRPα binds to a wildtype human CD47 with a dissociation constant (KD) of less than 100 nanomolar (nM), optionally from 1 nM to 100 nM, 1 nM to 75 nM, 1 nM to 50 nM, 1 nM to 25 nM, 1 nM to 10 nM, 10 nM to 100 nM, 10 nM to 75 nM, 10 nM to 50 nM, 10 nM to 25 nM, 25 nM to 100 nM, 25 nM to 75 nM, 25 nM to 50 nM, or 50 nM to 100 nM, 50 nM to 75 nM, or 75 nM to 100 nM. In various embodiments, the variant SIRPα binds to a wildtype human CD47 with a dissociation constant (KD) of less than 1 nM, optionally from 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 a wildtype human CD47 with a dissociation constant (KD) of less than 100 picomolar (pM). In various embodiments, the variant SIRPα binds to a wildtype human CD47 with a dissociation constant (KD) of from 1 pM to 100 pM, optionally from 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.

[0017] In various embodiments, 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 amino acid numbering of SEQ ID NO: 103 or SEQ ID NO:104. In various embodiments, at least one amino acid substitution is E54Q, corresponding to amino acid numbering of SEQ ID NO: 103 or SEQ ID NO:104.

[0018] In various embodiments, the one more amino acid substitutions comprise K53R, E54Q and S66T (L66T), corresponding to amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104. In various embodiments, 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 numbering of SEQ ID NO:103 or SEQ ID NO:104. In various embodiments, the one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I, corresponding to amino acid numbering of SEQ ID NO:103 or SEQ ID NO:104. In various embodiments, the first antigen binding domain comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:105. In various embodiments, the first antigen binding domain is set forth by an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:105. In various embodiments, the first antigen binding domain is or comprises the amino acid sequence set forth in SEQ ID NO: 105.

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

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

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

[0022] 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 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, an Fab, Fv, Fav, F(ab’)2, Fab’, dsFv, Fde, sdFv. In various embodiments, the second antigen binding domain is a single domain antibody that is a VHH. In various embodiments, the VHH is a camelid heavy chain antibody, a humanized VHH domain, an affinity maturated VHH domain, or a human VHH domain.

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

[0024] In various embodiments, the VHH domain binds to an IgV domain of a wild-type human SIRPα or a variant thereof. In various embodiments, the anti-SIRPα antibody or antigen- binding fragment is pan-reactive and binds to a wild-type SIRPα and at least one variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47. In various embodiments, the least one variant SIRPα comprises one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47. In various embodiments, the IgV domain of the one or more wild-type human SIRPα or variant thereof is selected from among: (i) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 103; (ii) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 104 ; (iii) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:105 ; (iv) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10 ; and (v) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 27.

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

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

[0027] In various embodiments, 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 numbering of SEQ ID NO:103 or SEQ ID NO:104. In various embodiments, the one or more amino acid substitutions are V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I, corresponding to amino acid numbering of SEQ ID NO:103 or SEQ ID NO:104. In various embodiments, the variant SIRPα is designated FB3, FD6, FA4 or CV1.

[0028] In various embodiments, the anti-SIRPα antibody or antigen-binding fragment binds a variant SIRPα, optionally the IgV domain of the variant SIRPα, comprising: (i) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 105; (ii) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10 ; or (iii) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 27.

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

[0030] In various embodiments, the linker is a substrate for a protease, optionally where the protease is an extracellular protease and / or the linker is a substrate for renin, pepsin C, napsin A, a matrix metalloprotease (MMP), matriptase, urokinase-type plasminogen activator (uPA), a disintegrin and metalloprotease (ADAM), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), legumain, urokinase, or hepsin.

[0031] In various embodiments, the proteolytically cleavable linker is a polypeptide that functions as a substrate for 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 among matriptase, a matrix metalloprotease (MMP), granzyme B, and combinations thereof. In various embodiments, the protease is matriptase. In various embodiments, the proteolytically cleavable linker is VHMPLGFLGPRQARVVN (SEQ ID NO:22). In various embodiments, the linker comprising the proteolytically cleavable linker comprises N- and / or C-terminal GS linker sequence. In various embodiments, the GS linker sequence is the sequence (GGGGS)n, wherein 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).

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

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

[0034] In various embodiments, the third antigen binding domain is a Fab. In various embodiments, the third antigen binding domain is a single chain antibody fragment. In various embodiments, the third antigen binding domain is a VHH. In various embodiments, the VHH is a camelid heavy chain antibody, a humanized VHH domain, an affinity maturated 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 twodifferent single chain antibody fragments. In various embodiments, the third antigen binding domain is biparatopic. In various embodiments, the first antigen binding domain, the second antigen binding domain, and / or the third antigen binding domain is an antibody. In various embodiments, the first antigen binding domain, the second antigen binding domain, and / or the third antigen binding domain includes an extracellular domain (ECD) of a cell surface-expressed protein, a fragment of the ECD of the cell surface-expressed protein, a mutated version (variant) of the ECD of the cell surface- expressed protein that is engineered to improve binding to target, a binding domain of an antibody, a functional fragment of an antibody, a variable domain thereof, a VH domain, a VL domain, a VNAR domain, a VHH domain, a single chain variable fragment (scFv), sc(Fv)2, an Fab, Fv, Fav, F(ab’)2, Fab’, dsFv, Fde, sdFv, a single domain antibody (sdAb), a nanobody, a bispecific antibody, a diabody, intrabody, domain antibody, antibody mimetic, zybody, polypeptide-Fc fusion, cameloid antibody, camelized antibody, masked antibody affybody, anti-idiotypic (anti-Id) antibody, single chain diabody, tandem diabody, VHH, anticalin, minibody, BiTE, ankyrin repeat protein, DARPIN, avimer, DART, TCR-like antibody, adnectin, affilin, trans-body, affibody, TrimerX, microprotein, fynomer, centyrin, KALBITOR, CAR, engineered TCR, or a functional fragment or a combination thereof. In various embodiments, the antibody includes an immunoglobulin constant domain selected from the group including or 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 a human immunoglobulin.

[0035] In various embodiments, a multispecific antigen binding construct further includes a fourth antigen binding domain that binds to a second target cell antigen, optionally where the second target cell antigen is expressed on the cell targeted for modulation (e.g., killing, e.g., by phagocytosis). In various embodiments, the fourth antigen binding domain is an antibody or antigen-binding fragment. In various embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv), sc(Fv)2, an 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 a 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 a target listed in Table 2.

[0036] In various embodiments, a multispecific antigen binding construct further includes 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 wildtype human IgG1 Fc region. In various embodiments, the immunoglobulin Fc region comprises an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO:98. In various embodiments, the immunoglobulin Fc region comprises the amino acid sequence set forth in SEQ ID NO:98.In various embodiments, the Fc region is a variant Fc region including one or more amino acid mutations or substitutions that increase the antibody dependent cellular cytotoxicity (ADCC)- promoting and / or antibody dependent cellular phagocytosis (ADCP)-promoting activity of the multispecific antigen-binding construct. In various embodiments, the variant Fc region comprises one or more amino acid mutations compared to a wildtype human IgG1 Fc region. In various embodiments, the variant Fc region includes a mutation of one or more positions selected from the group including or 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, 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 where one or more of the mutations are substitutions, further optionally where the substitutions are selected from the group including or 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, 267E 267L, 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, the one or more amino acid mutations are amino acid substitutions G236A, S239D and I332E. In various embodiments, the immunoglobulin Fc region comprises an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO:97. In various embodiments, the immunoglobulin Fc region comprises the amino acid sequence set forth in SEQ ID NO:97.

[0037] In various embodiments, the third antigen binding region is a 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 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 a second polypeptide comprising a light chain variable region(VL) and a light chain constant region (CL) of the Fab. In various embodiments, the third antigen binding region is a Fab and the multispecific antigen binding construct comprises: a first polypeptide comprising, in N- to C- terminal order, a heavy chain variable region (VH) and a 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 a second polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of the 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 a disulfide bond and wherein each of the second polypeptides are covalently linked to one of the first polypeptides by a disulfide bond. In various embodiments,

[0038] In various embodiments, the third antigen binding region is a Fab and the multispecific antigen binding construct comprises: a first polypeptide comprising, in N- to C-terminal order, a heavy chain variable region (VH) and heavy chain constant region (CH1) of the Fab, an immunoglobulin Fc region comprising the sequence of amino acids set forth in SEQ ID NO: 97, a first antigen binding domain comprising the sequence of amino acids set forth in SEQ ID NO: 10, a linker comprising the proteolytically cleavage linker set forth in SEQ ID NO: 12, and a second antigen binding domain comprising a sequence that has at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 13-21 and 28-36; and a second polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of the Fab. In various embodiments, the third antigen binding region is a Fab and the multispecific antigen binding construct comprises: a first polypeptide comprising, in N- to C-terminal order, a heavy chain variable region (VH) and heavy chain constant region (CH1) of the Fab, an immunoglobulin Fc region comprising the sequence of amino acids set forth in SEQ ID NO: 97, a first antigen binding domain comprising the sequence of amino acids set forth in SEQ ID NO: 27, a linker comprising the proteolytically cleavage linker set forth in SEQ ID NO: 12, and a second antigen binding domain comprising a sequence that has at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 13-21 and 28-36; and a second polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of the Fab.

[0039] In various embodiments, the second antigen binding domain comprises the sequence set forth in any one of SEQ ID NOS: 13-21 and 28-36. In various embodiments, wherein the multispecific antigen binding construct comprises a peptide linker between the immunoglobulin Fc region and the first antigen binding domain. In various embodiments, the peptide linker is a GS linker. In various embodiments, the GS linker is (GGGGS)n, wherein 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), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 24).

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

[0041] In various embodiments, the Fab is a Necitumumab Fab comprising a heavy chain comprising a sequence comprising at least 95% sequence identity to SEQ ID NO: 7 and a light chain comprising a least 95% sequence identity to SEQ ID NO: 2. In various embodiments, the Fab is a Necitumumab Fab comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2. In various embodiments, wherein the Fab is a Necitumumab Fab comprising a heavy chain comprising a sequence comprising at least 95% sequence identity to SEQ ID NO: 205 and a light chain comprising a least 95% sequence identity to SEQ ID NO: 2. In various embodiments, the Fab is a Necitumumab Fab comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 205 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0042] In various embodiments, the multispecifc polypeptide construct comprises a first polypeptide chain comprising a sequence comprising at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 110-120 and a second polypeptide chain comprising a sequence comprising at least 95% sequence identity to the sequence set forth in SEQ ID NO: 2. In variousembodiments, the multispecifc polypeptide construct comprises a first polypeptide chain comprising the sequence set forth in any one of SEQ ID NOS: 110-120 and a second polypeptide chain comprising the sequence set forth in SEQ ID NO: 2. In various embodiments, the multispecific polypeptide construct comprises a first polypeptide chain comprising a sequence comprising at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 125-137 and a second polypeptide chain comprising a sequence comprising at least 95% sequence identity to the sequence set forth in SEQ ID NO: 2. In various embodiments, the multispecifc polypeptide construct comprises a first polypeptide chain comprising the sequence set forth in any one of SEQ ID NOS: 125-137, and a second polypeptide chain comprising the sequence set forth in SEQ ID NO: 2.

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

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

[0045] In various embodiments, the first target cell antigen is EGFR. In various embodiments, the third antigen binding domain is an scFv derived from selected from the group consisting of Necitumumab (11F8), Cetuximab, Nimotuzumab and P2X. In various embodiments, the third antigen binding domain is an scFv derived from Necitumumab (11F8). In various embodiments, the scFv comprises a variable heavy (VH) chain comprising an amino acid sequence that has at least 95% sequence identity to the VH chain sequence present in SEQ ID NO: 207 and a variable light (VL) chain comprising an amino acid sequence that has at least 95% sequence identity to the VL chain sequence present in SEQ ID NO: 207. In various embodiments, the scFv comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:207. In various embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 207.

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

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

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

[0049] In various embodiments, at least one Fc polypeptide, optionally each Fc polypeptide, of the heterodimeric Fc region comprises at least one amino acid substitution to promote heterodimerization compared to a polypeptide of a homodimeric Fc region, optionally compared to an IgG1 Fc region. In various embodiments, the one or more amino acid substitution is a knob-into-hole modification or a charge mutation to increase electrostatic complementarity of the polypeptides. In various embodiments, the first Fc polypeptide of the heterodimeric Fc region comprises a amino acid substitution selected from among Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof and the second Fc polypeptide of the heterodimeric Fc region comprises the amino acid substitution T366W, and optionally wherein the first and second Fc polypeptides further comprises a amino acid substitution of a non-cysteine residue to a cysteine residue, wherein the amino acid substitution of thefirst polypeptide is at one of the position Ser354 and Y349 and the amino acid substitution of the second Fc polypeptide is at the other of the position Ser354 and Y349. In various embodiments, the first Fc polypeptide comprises amino acid substitutions Y349C, T366S, L368A, 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 set forth in SEQ ID NO: 100 and the second Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 101. In various embodiments, the first Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 106 and the second Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 107.

[0050] In various embodiments, the multispecific antigen binding construct comprises a first polypeptide comprising, in N- to C-terminal order, a heavy chain variable region (VH) and heavy chain constant region (CH1) of the Fab, a first immunoglobulin Fc region comprising the sequence of amino acids set forth in SEQ ID NO: 106, a first antigen binding domain comprising the sequence of amino acids set forth in SEQ ID NO: 27; a second polypeptide comprising n N- to C-terminal order, the VH and CH1 of the Fab, a second immunoglobulin Fc region comprising the sequence of amino acids set forth in SEQ ID NO: 107, a linker comprising the proteolytically cleavage linker set forth in SEQ ID NO: 12, and a second antigen binding domain comprising a sequence that has at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 13-21 and 28-36; and a third polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of the Fab. In various embodiments, the second antigen binding domain comprises the sequence set forth in any one of SEQ ID NOS: 13-21 and 28-36.

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

[0052] In various embodiments, the Fab is a Necitumumab Fab comprising a heavy chain comprising a sequence comprising at least 95% sequence identity to SEQ ID NO: 7 and a light chain comprising a least 95% sequence identity to SEQ ID NO: 2. In various embodiments, the Fab is a Necitumumab Fab comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2. In various embodiments, the Fab is a Necitumumab Fab comprising a heavy chain comprising a sequence comprising at least 95% sequence identity to SEQ ID NO: 205 and a light chain comprising a least 95% sequence identity to SEQ ID NO: 2. In various embodiments, wherein the Fab is a Necitumumab Fab comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 205 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0053] In at least certain aspects, the present disclosure provides a nucleic acid encoding a multispecific antigen binding construct according to the present disclosure. In at least certain aspects, the present disclosure provides an expression vector including a nucleic acid of the present disclosure. In at least certain aspects, the present disclosure provides a cell including an expression vector of the present disclosure. In at least certain aspects, the present disclosure provides a method for producing a multispecific antigen binding construct, the method including culturing the cell of the present disclosure, or a population of such cells, under conditions conducive for expression of the multispecific antigen binding construct from the expression vector by the cell. In various embodiments, a method for producing a multispecific antigen binding construct as provided herein further includes isolating the multispecific antigen binding construct from the cell or population of cells, or from the medium in which the cell or population of cells were cultured. In at least certain aspects, the present disclosure provides a pharmaceutical composition including the multispecific antigen binding construct of the present disclosure and a pharmaceutically acceptable carrier or excipient.

[0054] In at least certain aspects, the present disclosure provides a method for increasing modulation (e.g., killing, e.g., by phagocytosis) of a target cell by a myeloid cell, the method including: contacting, in the presence of a myeloid cell, a population of target cells with the multispecific antigen-binding construct of the present disclosure in an amount sufficient to modulate or increase modulation (e.g., killing, e.g., by phagocytosis) of the target cells by the myeloid cell. In various embodiments, the myeloid cell is a macrophage, a dendritic cell, a neutrophil, a tumorassociated macrophage (TAM), or a tumor infiltrating macrophage (TIM).. In various embodiments, the target cell is infected with a microbe or expresses a microbial antigen. In various embodiments, the microbial antigen is a viral antigen. In various embodiments, the cell is a cancer cell or the cell expresses a tumor associated antigen (TAA). In various embodiments, the TAA is selected from the group of TAAs listed in Table 2 or derived from a target listed in Table 2.

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

[0056] In at least certain aspects, the present disclosure provides a method for treating or delaying progression of an autoimmune or inflammatory disease in a subject, the method including administering to the subject an effective amount of a multispecific antigen-binding construct of the present disclosure, thereby treating and / or delaying the progression of the autoimmune or inflammatory disease in the subject. In various embodiments, the autoimmune or inflammatory disease is atherosclerosis, obesity, inflammatory bowel disease (IBD), Lyme disease, Hashimoto's thyroiditis, autoimmune uveitis, autoimmune valvular carditis, rheumatoid arthritis, allergic encephalitis, atopic skin disease, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylitis, type 1 diabetes mellitus, alopecia areata, vasculitis, and temporal arteritis, graft versus host disease (GVHD), asthma, COPD, eosinophilia, conjunctivitis, glomerular nephritis, autoimmune nephritis, a paraneoplastic autoimmune disease, cartilage inflammation, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenilereactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis , juvenile psoriatic arthritis, a fibrotic disease, juvenile Scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, enteropathic arthritis, reactive arthritis, Reiter's Syndrome, dermatomyositis, psoriatic arthritis, Scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodossa, Wegener's granulomatosis, arteritis, ploymyalgia rheumatica, sarcoidosis, Sclerosis, primary biliary Sclerosis, 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, rhinosinusitis, rhinosinusitis with polyps, eosinophilic esophogitis, eosinophilic bronchitis, Guillain-Barre disease, thyroiditis (e.g., Grave’s disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplantation rejection, kidney damage, hepatitis C-induced vasculitis, a viral infection, a bacterial infection, or spontaneous loss of pregnancy.

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

[0058] In at least certain aspects, the present disclosure provides a method for treating or delaying progression of a neurological disease in a subject, the method including administering to the subject an effective amount of a multispecific antigen-binding construct of the present disclosure, thereby treating and / or delaying the progression of the neurological disease in the subject. In various embodiments, the neurological disease is Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, an ophthalmic disorder, glaucoma, myotonic dystrophy, Guillain-Barre´ syndrome (GBS), Myasthenia Gravis, Bullous Pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson’s disease, traumatic brain injury (TBI), epilepsy, or Huntington’s disease (HD).

[0059] In various methods of the present disclosure, the subject is a mammal, e.g., where the mammal is a human, a non-human primate, a farm animal, a domestic animal, or a laboratory animal. In various methods of the present disclosure, the multispecific antigen-binding construct is administered orally, rectally, intravenously, intratumorally, or subcutaneously.

[0060] In at least certain aspects, the present 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 among SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, and 45; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from among SEQ ID NOs: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from among SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In various embodiments, the at least one SIRPα VHH domain comprises a CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 37, 46 and 61, respectively; SEQ ID NOs: 38, 46 and 61, respectively; SEQ ID NOs: 39, 47 and 62, respectively; SEQ ID NOs: 40, 48 and 63, respectively; SEQ ID NOs: 41, 49 and 64, respectively; SEQ ID NOs: 37, 50 and 61, respectively; SEQ ID NOs: 42, 51 and 65, respectively; SEQ ID NOs: 43, 52 and 66, respectively; SEQ ID NOs: 37, 53 and 67, respectively; SEQ ID NOs: 44, 54 and 68, respectively; SEQ ID NOs: 43, 55 and 63, respectively; SEQ ID NOs: 40, 56 and 69, respectively; SEQ ID NOs: 37, 57 and 70, respectively; SEQ ID NOs: 40, 55 and 63, respectively; SEQ ID NOs: 41, 58 and 71, respectively; SEQ ID NOs: 43, 59 and 72, respectively; SEQ ID NOs: 37, 60 and 73, respectively; or SEQ ID NOs: 45, 56 and 73, respectively. In various embodiments, the SIRPα is a human SIRPα. In various embodiments, the at least one SIRPα VHH domain comprises the sequence of amino acids set forth in any one of SEQ ID NOs:13-21 and 28-36 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 13-21 and 28-36, and binds SIRPα. In various embodiments, the at least one SIRPα VHH domain comprises the sequence of amino acids set forth in any one of SEQ ID NOs: 13-21 and 28-36.

[0061] In various embodiments, binding of the SIRPα VHH domain to SIRPα inhibits or reduces the binding of SIRPα to cluster of differentiation 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 an IgV domain of one or more wild-type human SIRPα or variant thereof. In various embodiments the IgV domain of the one or more wild- type human SIRPα or variant thereof is selected from among: (i) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 103; (ii) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:104; (iii) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:105; (iv) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10 ; and (v) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2.

[0062] In various embodiments, the VHH is pan-reactive and binds to a wild-type SIRPα and at least one variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47. In various embodiments, the VHH binds to (1) an IgV domain of a 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: (a) comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47; and / or (b) is a deglycosylated variant.

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

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

[0065] In at least certain aspects, the present disclosure provides a binding molecule comprising the SIRPα-binding molecule according to the present disclosure and a second binding domain that binds to a second antigen. In various embodiments, the second antigen is a tumor antigen. In various embodiments, the tumor antigen is a tumor associated antigen (TAA) selected from the group of TAAs listed in Table 2 or derived from a target 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 that binds to a second antigen is an antibody or antigen-binding fragment.

[0066] In at least certain aspects, the present disclosure provides a nucleic acid encoding the SIRPα-binding molecule according to the present disclosure. In at least certain aspects, the present disclosure provides an expression vector including a nucleic acid of the present disclosure. In at least certain aspects, the present disclosure provides a cell including an expression vector of the present disclosure. In at least certain aspects, the present disclosure provides a method for producing a SIRPα- binding molecule, the method including culturing the cell of the present disclosure, or a population of such cells, under conditions conducive for expression of the SIRPα-binding molecule, from the expression vector by the cell. In various embodiments, a method for producing a SIRPα-binding molecule, as provided herein further includes isolating the SIRPα-binding molecule, from the cell or population of cells, or from the medium in which the cell or population of cells were cultured. In at least certain aspects, the present disclosure provides a pharmaceutical composition including the SIRPα-binding molecule of the present disclosure and a pharmaceutically acceptable carrier or excipient.

[0067] In at least certain aspects, the present disclosure provides a method for treating or delaying progression of a cancer in a subject, the method including administering to the subject an effective amount of a SIRPα-binding molecule, binding molecule, or pharmaceutical composition of the present disclosure, thereby treating and / or delaying the progression of the cancer in the subject. In various embodiments, the cancer is an adenocarcinoma, a bile duct (biliary) cancer, a bladder cancer, a bone cancer, a breast cancer, a triple-negative breast cancer, a Her2-negative breast cancer, a carcinoid cancer, a cervical cancer, a cholangiocarcinoma, a colorectal cancer, a colon cancer, an endometrial cancer, an esophageal cancer, a glioma, a head and neck cancer, a head and neck squamous cell cancer, a leukemia, a liver cancer, a lung cancer, a non-small cell lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a metastatic castration-resistant prostate carcinoma, a renal cancer, a sarcoma, a skin cancer, a squamous cell cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, or a urothelial cancer. In various embodiments, the method further comprisesadministering to the subject an additional therapeutic agent for treating the cancer. In various embodiments, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG.1A shows depictions of six anti-EGFR antibodies. The constructs included: Necitumumab (11F8) hIgG1 (C-1), Necitumumab (11F8) hIgG1 EEF (C-2), Cetuximab hIgG1 EEF (C-3), Nimotuzumab hIgG1 EEF (C-4), P2X hIgG1 EEF (C-5), and 11F8 scFv hIgG1 EEF (C-6). The 11F8 scFv hIgG1 EEF construct (C-6) included G44HC / G100LC scFv stabilizing mutations. EEF = enhanced effector function (via G236A / S239D / I332E mutations in the FC, represented by dots)

[0069] FIG.1B shows results from a flow cytometry assay to assess EGFR cell binding of the anti-EGFR antibodies on A431 (high EGFR) and MCF7 (low EGFR) cells. Results are shown as mean fluorescence intensity (MFI).

[0070] FIG.2A shows depictions of four anti-EGFR-SIRPα fusion constructs that contained the EGFR antibody Necitumumab (11F8) hIgG1 EEF or the 11F8 scFv IgG1 EEF, and domain 1 of SIRPα (SIRPα-D1). The position of the SIRPα was varied across the constructs. For the 11F8 scFv IgG1 EEF- SIRPα fusion construct, the SIRPα-D1 was fused to the C-terminal of the Fc (C-7). For the Necitumumab (11F8) hIgG1 EEF - SIRPα fusion constructs, the SIRPα-D1 was fused to the C- terminal of the heavy chain (fusion 1, C-8), the C-terminal of the light chain (2, C-9), or the N- terminal 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) included G44HC / G100LC scFv stabilizing mutations. EEF = enhanced effector function (via G236A / S239D / I332E mutations in the FC, represented by dots).

[0071] FIG.2B shows results from a flow cytometry assay to assess CD47 cell binding of the anti-EGFR-SIRPα fusion constructs on MCF7 WT cells (CD47+ / EGFR-) and MCF7 CD47 knock out cells (KO; CD47- / EGFR-). Results are shown as mean fluorescence intensity (MFI). Necitumumab hIgG1 EEF (C-2) and 11F8 scFv hIgG1 EEF without SIRPα (C-6) were used as negative controls for CD47 binding.

[0072] FIG.3A shows depictions of an anti-EGFR biparatopic VHH hIgG1 EEF SIRPα fusion construct (C-64) and a control construct without the SIRPα fusion (C-63). EEF = enhanced effector function (via G236A / S239D / I332E mutations in the FC, represented by dots).

[0073] FIG.3B shows results from a flow cytometry assay to assess CD47 cell binding of the anti-EGFR biparatopic VHH hIgG1 EEF SIRPα fusion (C-64) on MCF7 WT cells (CD47+ / EGFR-) and MCF7 CD47 knock out cells (KO; CD47- / EGFR-). Results are shown as mean fluorescence intensity (MFI). Necitumumab hIgG1 EEF (C-2) and the anti-EGFR biparatopic VHH hIgG1 EEF construct without SIRPα (C-63) were used as negative controls for CD47 binding.

[0074] FIG.3C shows results from a flow cytometry assay to assess EGFR cell binding of the anti-EGFR biparatopic 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). Necitumumab hIgG1 EEF (C-2) and the anti-EGFR biparatopic VHH hIgG1 EEF construct without SIRPα (C-63) were used as positive controls for EGFR binding.

[0075] FIG.4A shows depictions of anti-EGFR-anti-CD47 fusion constructs that contained the EGFR antibody Necitumumab (11F8) hIgG1 EEF and an anti-CD47 VHH. antibody. The position of the anti-CD47 VHH was varied across the constructs: an anti-CD47 VHH antibody was fused C-terminal to the heavy chain (fusion 1, C-11), C-terminal to the light chain (fusion 2, C-12), or N-terminal to the light chain (fusion 3, C-13). The C-terminal light chain fusion construct (C-12) included G44HC / G100LC scFv stabilizing mutations. EEF = enhanced effector function (via G236A / S239D / I332E mutations in the FC, represented by dots).

[0076] FIG.4B shows results from a flow cytometry assay to assess CD47 cell binding of the Necitumumab-anti-CD47 VHH fusions on MCF7 WT cells (CD47+ / EGFR-) and MCF7 CD47 knock out cells (KO; CD47- / EGFR-). Results are shown as mean fluorescence intensity (MFI). Necitumumab hIgG1 EEF (C-2) was used as a negative control for CD47 binding.

[0077] FIG.5A shows an exemplary workflow for the phagocytosis screen of the multi- specific antigen constructs. THP-1 monocytes stably expressing nuclear-restricted tag GFP2 were used as effector cells and A431 human epidermoid carcinoma cells labeled with labeled with pHrodo Orange were used as target cells. Cells were incubated with the multi-specific antigen constructs and imaged using phase, green, and orange channels every 45 minutes for 12 hours. Phagocytosed cells were defined as green+high-orange overlapping, as pHrodo only fluoresces at low pH (upon phagocytosis).

[0078] FIG.5B shows the amount of phagocytosis after addition of each fusion construct (C-1-C-15 and C-64-C-64) at a concentration of 50 nM. Results are shown as area under the curve (AUC).

[0079] FIG.5C shows the amount of phagocytosis after addition of Necitumumab (11F8) hIgG1 EEF SIRPα fusion constructs (C-8, C-9, and C-10) at concentrations of 0.5, 5, and 50 nM. Results are shown as area under the curve (AUC). The Necitumumab (11F8) hIgG1 (C1) construct and Necitumumab (11F8) hIgG1 EEF (C2) construct were used as non-SIRPα controls. A control lacking an anti-EGFR domain, in which SIRPα was fused to the C-terminus of a homodimeric Fc IgG1 EFF polypeptide (C-14, CD47 blocker only), was also used alone or in combination with Necitumumab (11F8) hIgG1 EEF (C2, anti-EGFR only).

[0080] FIG.5D shows the amount of phagocytosis after addition of a CD47 blocker only (C14, Fc IgG1 EFF-SIRPα) construct, tumor targeting only construct (Necitumumab (11F8) hIgG1 EEF; C2), a combination of CD47 blocker (C14) + anti-EGFR (C2) constructs, or as a multi-specificfusion construct (Necitumumab (11F8) hIgG1 EEF SIRPα fusion 1; C-8) at a concentration of 0.5 nM. Results are shown as fold change in phagocytosis. Asterisks denote a P<0.0001, as determined from a two-way ANOVA with Tukeys multiple comparisons test.

[0081] FIG.6A shows depictions of CD47 blocker only (no-anti-EGFR) constructs in which the SIRPα D1 was fused to either the C-terminus (SIRPα D1 Fc fusion 1, C-14) or the N-terminus (SIRPα D1 Fc fusion 2, C-15) of a homodimeric Fc IgG1 EFF polypeptide. Also depicted are constructs where a CD47 extracellular domain was used as a “mask domain” to block interaction of a SIRPα-D1 with cell surface CD47. The CD47 extracellular domain and the SIRPα-D1 were each linked to one of the polypeptide chains of a heterodimeric Fc with knob-in-hole mutations either at the N-terminus (KiH Fc EEF CD47 and SIRPα fusion 1, C-16M) or at the C-terminus (KiH Fc EEF CD47 and SIRPα fusion 2, C-17M) of each Fc polypeptide. The mask domain was linked to the Fc region by a cleavable linker to conditionally control the interaction between the CD47 extracellular domain and the SIRPα D1.

[0082] FIG.6B shows results from a flow cytometry assay to assess CD47 cell binding of the SIRPαD1-Fc fusions with or without a mask domain (C14, C15, C16M and C17M) on 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).

[0083] FIG.7A shows depictions of three formats for anti-EGFR-SIRPα-mask fusion constructs. The formats included: Necitumumab (11F8) hIgG1 EEF:SIRPα-WT:mask fusion (Format #1), Necitumumab (11F8) hIgG1 EEF:SIRPα-CV1:mask fusion (Format #2), and an asymmetric variant KiH Necitumumab (11F8) hIgG1 EEF where the hole arm was fused to SIRPα-CV1 and the knob arm was fused to a mask (Format #3).

[0084] FIG.7B shows results from a flow cytometry assay to assess CD47 cell binding of anti-EGFR-SIRPα-mask fusion constructs on MCF7 WT (CD47+ / EGFR-) and MCF7 KO (CD47- / EGFR+) cells. The fusion constructs were assayed with or without digestion with matriptase at concentrations from 100 nM to 0.6 pM. Exemplary results are shown for constructs: without a mask (C-50), with a mask+protease (C-55M Digested), with a mask (C-55M Undigested), and with a mask with a non-cleavable linker (C61-M). Results are shown as median fluorescence intensities (MFI) plotted against the concentrations of the fusion constructs. DETAILED DESCRIPTION

[0085] The present disclosure relates to compositions and methods useful for the treatment of disease (e.g., cancer). In particular, the present disclosure provides compositions that can selectively direct myeloid cell activity to target cells (e.g., direct killing and / or indirect killing by myeloid cells directed toward target cells), such as by being selectively activated in particular context of interest, such as an activating tumor microenvironment. Among provided embodiments is amultispecific binding molecule platform that is selectively activated in tumors to promote macrophage killing of tumor cells. The platform includes various format configurations that combine for targeting to the tumor, priming with a masking domain for conditional activation to block anti-phagocytic protein activity in a tumor-selective manner, and promoting macrophage dependent antibody- dependent cellular cytoxicity (ADCC) and phagocytosis (ADCP) to promote tumor cell killing. In some embodiments, a therapeutic binding agent encompassed by the present disclosure can selectively direct elimination of target cells by antibody-dependent cellular phagocytosis (ADCP). In some embodiments, a therapeutic binding agent encompassed by the present disclosure can selectively direct elimination of target cells by antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, a therapeutic binding agent encompassed by the present disclosure can selectively direct elimination of target cells by direct killing.

[0086] There is a need for improved therapeutics that are able to promote the potential killing activity of myeloid cells, including macrophages. Macrophages make up up to 50% of all cells in the solid tumor (Mellman et al, Immunity (2023); Cheng et al. J. Hematology & Oncology (2023)). However, existing therapies have not yet been effective to exploit macrophages for direct tumor killing. For example, the immunosuppressive tumor microenvironment created by macrophages is a major barrier to immune-oncology success. Although macrophages are capable of eating and killing tumor cells, they are often deactivated and immunosuppressive in the tumor microenvironment. For instance, many tumor cells and cells in the tumor microenvironment express CD47 and suppress the phagocytic activity of macrophages. According the therapeutic potential of macrophages is not being fully realized, including the potential of macrophages to promote rapid innate immunity-driven tumor debulking and bridging innate and adaptive immunity for long-lasting clinical responses. Thus, there is a great need to unlock macrophages for direct tumor killing including as part of an orchestrated immune attack that can achieve durable responses.

[0087] In some embodiments, a therapeutic binding agent encompassed by the present disclosure can include: (1) at least one binding domain that binds an anti-phagocytic protein (APP) (e.g., as expressed on myeloid cells or cells targeted for modulation (e.g., direct killing and / or indirect killing)) (APP-binding domain), (2) at least one binding domain that conditionally inhibits interaction of the anti-APP binding domain with its target (mask domain), and (3) a proteolytically cleavable linker positioned such that its cleavage relieves the inhibition of APP binding by the mask domain, thereby achieving activatable activity. Therapeutic binding agents encompassed by the present disclosure can comprise one or more additional elements, such as, for example 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 instance, the provided binding agents are multispecific binding agents that further include an immunoglobulin Fc region for activating an FcγR. In some embodiments, the immunoglobulin Fc region includes Fc-enhancing mutations that are further known to enhance macrophage activity to promote macrophage dependent ADCC / ADCP. Among the Fc-enhancing mutations are those that have increased affinity to FcγRIIIa (e.g., S239D and I332E) and greater affinity for FcγRIIa relative to the inhibitory receptor FcγRIIb (e.g., G236A). For instance, the Fc-enhancing triple mutant S239D, I332E and G236A can improve the ratio of RIIa to RIIb binding while maintaining increased affinity to FcγRIIIa. These elements, therapeutic binding agents that include one or more of such elements, and uses thereof, e.g., for treatment of cancer, are further described herein.

[0088] In embodiments of provided binding agents, the APP is CD47 and the binding agent includes at least one binding domain to bind CD47 to block the suppression of macrophages and other myeloid cells. The CD47 blockade can induce long-term anti-tumor immunity and also bridge the innate and adaptive immune systems. Moreover, combining the CD47 blockade with a binding agent that allows for FcγR interaction can further promote myeloid-directed anti-tumor response including to distant tumors. Further, the provided binding agents are conditionally activated to conditionally control binding the APP specifically in the tumor microenvironment and not in tumor tissues using a masking domain that can be released in the tumor, thereby maximizing selective activation in tumors to promote macrophage killing of tumor cells specifically and not normal cells. For instance, in provided embodiments, tumor selectivity can be achieved by protease-mediated activation of a proteolytically cleavable linker that joins the masking domain to the APP binding domain, thereby selectively activating the provided binding agent in the tumor. This addresses shortcomings in some existing CD47 blockade therapies since CD47 is ubiquitously present in both normal and tumor cells. Off-tumor binding to CD47 can lead to toxicity and poor pharmacology including anemia, thrombocytopenia and tissue sink (decreased availability), whereas on-target binding specific to the tumor is expected to lead to anti-tumoral activity and promote ADCC / ADCP to promote tumor cell killing. The provided approach also has the flexibility to be tunable by using a SIRPα binding domain as the binding domain that binds to the CD47 APP, including a wild-type SIRPα and various engineered variants with intermediate or high affinities. The provided approach provides for a unique therapeutic that allows for enhanced Fc function and CD47 blockade without other compromises such as silencing the Fc or using a low affinity CD47 blocker.

[0089] The present disclosure further relates to functional equivalents of the APP-binding domain (i.e., APP-binding domain means), mask domain (i.e., mask domain means), proteolytically cleavable linker (i.e., proteolytically cleavable linker means), myeloid promoting and / or activating element (i.e., myeloid promoting means and / or myeloid activating means), and / or target cell-binding domain (i.e., target cell-binding domain means). Functional equivalents of each respective element would differ insubstantially from the respective element as described herein due to performing substantially the same function as the respective element in substantially the same way to obtain substantially the same result. The functional equivalents of each respective 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, immunecell proliferation, and / or immune cell migration, representative examples of which functions, elements, and / or equivalents are further described herein.

[0090] The present disclosure is based, at least in part, on the recognition that cells associated with certain diseases, conditions, or disorders (which terms can be used interchangeably herein), such as cancer, can evade phagocytosis through aberrant and / or increased expression of APPs, contributing to pathology. Increased expression of APPs (e.g., CD47) inhibits phagocytosis and correlates with poor prognosis. Binding agents provided herein conditionally (activatably) direct myeloid cell activity to such cells, thereby treating disease.

[0091] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0092] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. DEFINITIONS

[0093] A, An, The, Or: As used herein, “a”, “an”, and “the” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” discloses embodiments of exactly one element and embodiments including more than one element. As used herein, the terms “or” and “and / or”, as conjunctions in a list of at least two elements, encompass and disclose embodiments in which the listed elements are included in the alternative, together, or in any combination.

[0094] About: The term “about,” in some embodiments, encompasses values that are within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, inclusive, or any range in between (e.g., plus or minus 2%-6%), of a value that is measured. In some embodiments, the term “about” refers to the inherent variation of error in a method, assay, or measured value, such as the variation that exists among experiments.

[0095] Affinity: As used herein, “affinity” refers to the strength of the sum total of non- covalent interactions between a particular binding agent (e.g., an antigen-binding agent), and / or a binding site thereof, with a binding target (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to a 1:1 interaction between a binding agent and a binding target thereof (e.g., an antibody with an antigen target of the antibody). Those of skill in the art appreciate that a change in affinity can be described by comparison to a reference (e.g., increased or decreased relative to a reference), or can be described numerically. Affinity can be measured and / or expressedin a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD) and / or equilibrium association constant (KA). The KDis the quotient of koff / kon, whereas KAis the quotient of kon / koff, where konrefers to the association rate constant of, e.g., antibody with antigen, and koffrefers to the dissociation of, e.g., antibody from antigen. konand koffcan be determined by techniques known to those of skill in the art, such as BIACORE®or KinExA.

[0096] Agent: As used herein, the term “agent” may refer to any chemical entity, including without limitation any of one or more of an atom, molecule, compound, amino acid, polypeptide, nucleotide, nucleic acid, protein, protein complex, liquid, solution, saccharide, polysaccharide, lipid, or combination or complex thereof.

[0097] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and / or one or more CDRs). Thus, the term antibody includes, without limitation, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents including the same. Antibodies can be naturally occurring immunoglobulins (e.g., generated by an organism reacting to an antigen). Synthetic, non-naturally occurring, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.

[0098] As is well known in the art, typical human immunoglobulins are approximately 150 kD tetrameric agents that include two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other to form a structure commonly referred to as a “Y-shaped” structure. Typically, each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain includes three CH domains: CH1, CH2 and CH3. A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.” Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of a heavy and / or a light chain are typically understood to provide a binding moiety that can interact with an antigen. Constant domains can mediate binding of an antibody to various immune system cells (e.g., effector cells and / or cells that mediate cytotoxicity), receptors, and elements of the complement system. Heavy and light chains are linked to one another by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to oneanother, so that the dimers are connected to one another and the tetramer is formed. When natural immunoglobulins fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure.

[0099] In some embodiments, an antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody). In some embodiments, an antibody includes 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 that includes two heavy chain monomers and two light chain monomers. Moreover, the term “antibody” can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and / or functional features including without limitation intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies® minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies,, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, KALBITOR®s, CARs, engineered TCRs, and antigen-binding fragments of any of the above.

[0100] Single domain antibody: As used herein, the term “single domain antibody,” which may be interchangeably referred to as “VHH domain” or “heavy chain only antibody variable domain,” refers to a single chain antigen binding domain that is capable of binding to an antigen or epitope, independently of a second light chain variable domain. Single domain antibody VHH are distinguished from variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH domains” or “VH regions” ) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains” or “VL domains”) . A VHH domain may be a human domain, but also includes a single domain from other species such as rodent, nurse shark and Camelid VHH domains. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanized according to standard techniques available in the art and are considered as “single domain antibodies” . VHH includes camelid VHH domains (including those derived from species including camel, llama, alpaca, dromedary, and guanaco) and humanized VHH domains. Such VHH domains may be humanized according to standard techniques available in the art. VHH domains also include synthetic VHHdomains including human-like VHH domains such as those in which amino acids at each of positions 44 and 45 or 37, 44, 45 and 47, based on Kabat numbering, somprise amino acids at the corresponding positions of a camelid VHH (see e.g., PCT publication No. WO2021 / 178263).

[0101] A “humanized” antibody refers to an antibody (e.g. a VHH) comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, all or substantially all of the CDRs of a humanized VHH correspond to those of a non-human VHH, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

[0102] An “affinity-matured” antibody, such as a VHH, has one or more alterations in one or more CDRs which result in an improved affinity for an antigen, as compared to the respective parental albumin binding molecule. Affinity-matured albumin binding molecules of the invention may be prepared by methods known in the art, for example, as described by KS Johnson and RE Hawkins, “Affinity maturation of antibodies using phage display” , Oxford University Press 1996.

[0103] In some embodiments, an antibody includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or variable domain. In some embodiments, an antibody can be a covalently modified (“conjugated”) antibody (e.g., an antibody that includes a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art.

[0104] An antibody including a heavy chain constant domain can be, without limitation, an antibody of any known class, including but not limited to, IgA, secretory IgA, IgG, IgE and IgM, based on heavy chain constant domain amino acid sequence (e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (µ)). IgG subclasses are also well known to those in the art and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. As used herein, a “light chain” can be of a distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the light chain constant domain. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human immunoglobulins. Naturally-produced immunoglobulins are glycosylated, typically on the CH2 domain. As is known in the art, affinity and / or other binding attributes of Fc domains (which can be interchangeably referred to as “Fc regions”) for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, antibodies produced and / or utilized inaccordance with the present disclosure include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation.

[0105] Antibody-dependent cell-mediated cytotoxicity (ADCC): The term refers to a form of cytotoxicity in which secreted antibodies bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No.5,500,362 or 5,821,337, may be performed. As is well-known in the art, the Fc portions may be engineered to effect a desired interaction or lack thereof with Fc receptors.

[0106] Antibody fragment: As used herein, an “antibody fragment” refers to antibody fragment or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof. An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be recombinantly produced (i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced. In some embodiments, an antibody fragment (particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.

[0107] Antigen-Binding Domain: The term “antigen-binding domain” or “binding portion” refers to the part of a binding molecule, such as an immunoglobulin molecule, that participates in antigen binding. For a conventional four-chain antibody, or of a Fab fragment, a F (ab') 2 fragment, an Fv fragment such as a disulfide linked Fv or a scFv fragment, or a diabody or other antibody fragment derived from a conventional four-chain antibody, the antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Iin these cases, binding to the respective epitope of an antigen occurs by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e. by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus, the term “FR” refers to amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed 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 of a bound antigen, andthe three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989). For a single domain antibody or VHH, the antigen-binding domain includes the heavy chain variable domain that includes the three CDRs of the heavy chain variable domain. VHH domains can specifically bind to an epitope without an additional antigen binding domain (as opposed to VH or VL domains in a conventional 4-chain antibody, in which case the epitope is recognized by a VL domain together with a VH domain). Many proteins also include immunoglobulin domains, known as immunoglobulin-like (Ig-like) domains, that are regions of proteins that are homologous to the V or C domains of immunoglobulin protein responsible for binding antigens in immunoglobulins. These immunoglobulin domains in proteins of the immunoglobulin superfamily are classified as IgV or IgC domains and can serve as antigen binding domain that participate in antigen binding.Associated with: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, or a combination thereof.

[0108] Binding: As used herein, the term “binding” refers to a non-covalent association between or among two or more agents. “Direct” binding involves physical contact between agents; indirect binding involves physical interaction by way of physical contact with one or more intermediate agents. Binding between two or more agents can occur and / or be assessed in any of a variety of contexts, including where interacting agents are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier agents and / or in a biological system or cell).

[0109] The terms “specific binding” or “specifically binds” refer to the non-covalent interactions of the type that occur between binding partners, such as between a receptor and its ligand or an immunoglobulin molecule and an antigen, for which the binding interaction is specific or of a high affinity. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (KD) of the interaction, wherein a smaller KDrepresents a greateraffinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of Koff / Kon enables the cancellation of all parameters not related to affinity and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present disclosure is said to specifically bind to an antigen (e.g., EGFR), when the binding constant (Kd) is ≤1 µM, for example, in some embodiments ≤ 100 nM, in some embodiments ≤ 10 nM, and in some embodiments ≤ 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0110] A protein (such as a protein, immunoglobulin, an antibody, an immunoglobulin single variable domain) that can “bind to" or “specifically bind to” , that “has affinity to” and / or that “has specificity for” a certain epitope, antigen or protein is said to be “against” or “directed against” said epitope, antigen or protein or is a “binding” molecule with respect to such epitope, antigen or protein.

[0111] Cancer: As used herein, the term “cancer” refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they display an abnormally elevated proliferation rate and / or aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a cancer can include one or more tumors. In some embodiments, a cancer can be or include cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a cancer can be or include a solid tumor. In some embodiments, a cancer can be or include a hematologic tumor.

[0112] Chemotherapeutic agent: As used herein, the term “chemotherapeutic agent,” consistent with its use in the art, refers to one or more agents known, or having characteristics known to, treat or contribute to the treatment of cancer. In particular, chemotherapeutic agents include pro- apoptotic, cytostatic, and / or cytotoxic agents. In some embodiments, a chemotherapeutic agent can be or include alkylating agents, anthracyclines, cytoskeletal disruptors (e.g. microtubule targeting moieties such as taxanes, maytansine, and analogs thereof, of), epothilones, histone deacetylase inhibitors HDACs), topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), kinase inhibitors, nucleotide analogs or nucleotide precursor analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids, and / or analogs that share a relevant anti- proliferative activity. In some particular embodiments, a chemotherapeutic agent can be or include of Actinomycin, All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin,Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and / or analogs thereof (e.g. DM1) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, or a combination thereof. In some embodiments, a chemotherapeutic agent can be utilized in the context of an antibody-drug conjugate. In some embodiments, a chemotherapeutic agent is one found in an antibody-drug conjugate selected from hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1- SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4- Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG- 5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vorsetuzumab mafodotin, and lorvotuzumab mertansine. In some embodiments, a chemotherapeutic agent can be or include of farnesyl-thiosalicylic acid (FTS), 4-(4-Chloro-2-methylphenoxy)-N- hydroxybutanamide (CMH), estradiol (E2), tetramethoxystilbene (TMS), δ-tocatrienol, salinomycin, or curcumin.

[0113] Complement dependent cytotoxicity (CDC): The term refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to antibodies which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano- Santoro et al. (1997), may be performed.

[0114] Domain: The term “domain” as used herein refers to a section or portion of an entity. In some embodiments, a “domain” is associated with a particular structural and / or functional feature of the entity so that, when the domain is physically separated from the rest of its parent entity, it substantially or entirely retains the particular structural and / or functional feature. Alternatively or additionally, a domain may be or include a portion of an entity that, when separated from that (parent) entity and linked with a different (recipient) entity, substantially retains and / or imparts on the recipient entity one or more structural and / or functional features that characterized it in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide; in some such embodiments, a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, α-helix character, β-sheet character, coiled- coil character, random coil character, etc.), and / or by a particular functional feature (e.g., bindingactivity, enzymatic activity, folding activity, signaling activity, etc.). In some embodiments, a domain is or includes a characteristic portion or characteristic sequence element.

[0115] Environment or microenvironment: The terms “environment” and “microenvironment” generally refer to the localized area in a tissue area of interest, or characteristics thereof, and may, for example, refer to a “tumor microenvironment.” The term “tumor microenvironment” or “TME” refers to the surrounding microenvironment that constantly interacts with tumor cells which is conducive to allow cross-talk between tumor cells and its environment. The tumor microenvironment may include the cellular environment of the tumor, surrounding blood vessels, immune cells, fibroblasts, bone marrow derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix. The tumor environment may include tumor cells or malignant cells that are aided and influenced by the tumor microenvironment to ensure growth and survival. The tumor microenvironment may also include tumor-infiltrating immune cells, such as lymphoid and myeloid cells, which may stimulate or inhibit the antitumor immune response, and stromal cells such as tumor-associated fibroblasts and endothelial cells that contribute to the tumor's structural integrity. Stromal cells may include cells that make up tumor-associated blood vessels, such as endothelial cells and pericytes, which are cells that contribute to structural integrity (fibroblasts), as well as tumor- associated macrophages (TAMs) and infiltrating immune cells, including monocytes, neutrophils (PMN), dendritic cells (DCs), T and B cells, mast cells, and natural killer (NK) cells. The stromal cells make up the bulk of tumor cellularity, while the dominating cell type in solid tumors is the macrophage.

[0116] Fragment: As used herein, “fragment” refers a structure that is or includes a discrete portion of a reference agent (sometimes referred to as the “parent” agent). In some embodiments, a fragment lacks one or more moieties found in the reference agent. In some embodiments, a fragment is or includes one or more moieties found 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, a fragment of a 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 found in the reference molecule.

[0117] In some embodiments, the reference agent is a polymer such as a polynucleotide or polypeptide. In some embodiments, a fragment of a 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 monomeric units (e.g., residues) of the reference polymer. In some embodiments, a fragment of a polymer 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 monomeric units (e.g., residues) found in the reference polymer. A fragment of areference polymer is not necessarily identical to a corresponding portion of the reference polymer. For example, a fragment of a reference polymer can be a polymer having a sequence of residues 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 identity to the reference polymer. A fragment may, or may not, be generated by physical fragmentation of a reference agent. In some instances a fragment is generated by physical fragmentation of a reference agent. In some instances, a fragment is not generated by physical fragmentation of a reference agent and can be instead, for example, produced by de novo synthesis or other means.

[0118] Improve, increase, inhibit, decrease or reduce: As used herein, the terms “improve”, “increase”, “inhibit”, “decrease” and “reduce”, and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference.

[0119] Inhibit or downregulate: The term “inhibit” or “downregulate” includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. In some embodiments, cancer is “inhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented. Similarly, a biological function, such as the function of a protein, is inhibited if it is decreased as compared to a reference state, such as a control like a wild-type state. Such inhibition or deficiency may be induced, such as by application of an agent at a particular time and / or place, or may be constitutive, such as by a heritable mutation. Such inhibition or deficiency may also be partial or complete (e.g., essentially no measurable activity in comparison to a reference state, such as a control like a wild-type state). In some embodiments, essentially complete inhibition or deficiency is referred to as “blocked.” In one embodiment, the term refers to reducing the level of a given output or parameter to a quantity (e.g., background staining, biomarker signaling,biomarker immunoinhibitory function, and the like) which is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less than the quantity in a corresponding control. A reduced level of a given output or parameter need not, although it may, mean an absolute absence of the output or parameter. The present disclosure does not require, and is not limited to, methods that wholly eliminate the output or parameter. The given output or parameter may be determined using methods well-known in the art, including, without limitation, immunohistochemical, molecular biological, cell biological, clinical, and biochemical assays, as discussed herein. The term “promote” or “upregulate” has the opposite meaning.

[0120] Linker: As used herein, “linker” is used to refer to that portion of a multi-element agent that connects different elements to one another. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide including a linker element has an overall structure of the generalform S1-L-S2, wherein S1 and S2 may be the same or different and represent two domains associated with one another by the linker. In some embodiments, a polypeptide 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 length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121- 1123).

[0121] Modulate: The term “modulate” and its grammatical equivalents refer to and encompass either or both of increasing and decreasing.

[0122] Operably linked: As used herein, “operably linked” refers to the association of at least a first element and a second element such that the component elements are in a relationship permitting them to function in their intended manner. For example, a nucleic acid sequence or amino acid sequence is operably linked with another sequence if it modifies the expression, structure, or activity of the linked sequence, e.g., in an 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 encoded polypeptide. In many cases, two amino acid sequences are operably linked if they are expressed as a single polypeptide.

[0123] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable,” as applied to one or more, or all, component(s) for formulation of a composition as disclosed herein, means that each component must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0124] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that facilitates formulation of an agent (e.g., a pharmaceutical agent), modifies bioavailability of an agent, or facilitates transport of an agent from one organ or portion of a subject to another. Some examples of materials which can serve 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; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions;polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0125] Pharmaceutical composition or formulation: As used herein, the term “pharmaceutical composition” or “formulation” refers to a composition in which a therapeutic agent is formulated together with one or more pharmaceutically acceptable carriers.

[0126] Reference: As used herein, “reference” refers to a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual, or population thereof, or a measure or characteristic representative thereof, is compared with a reference, an agent, sample, sequence, subject, animal, or individual, or population thereof, or a measure or characteristic representative thereof. In some embodiments, a reference is a measured value. In some embodiments, a reference is an established standard or expected value. In some embodiments, a reference is a historical reference. A reference can be quantitative of qualitative. Typically, as would be understood by those of skill in the art, a reference and the value to which it is compared represent assessments under comparable conditions. Those of skill in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison. In some embodiments, an appropriate reference may be an agent, sample, sequence, subject, animal, or individual, or population thereof, under conditions those of skill in the art will recognize as comparable, e.g., for the purpose of assessing one or more particular variables (e.g., presence or absence of an agent or condition), or a measure or characteristic representative thereof.

[0127] Small molecule: The term “small molecule” is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which may be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non- peptidic compounds. The term is intended to encompass all stereoisomers, geometric isomers, tautomers, and isotopes of a chemical structure of interest, unless otherwise indicated.

[0128] Therapeutic agent: As used herein, the term “therapeutic agent” refers to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be that of a model organism (e.g., an animal model, humanized animal model, animal model comprising a human immune system, and the like, of a disorder of interest, such as a cancer) or a human population. In some embodiments, an appropriate population can be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, atherapeutic agent is a substance that can be used for treatment of a disease, disorder, or condition. In some embodiments, a therapeutic agent is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent for which a medical prescription is required for administration to humans.

[0129] Subject: As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human, rat, or mouse). In particular embodiments, a subject is a human. In some embodiments, a subject is suffering from a disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject is not suffering from a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a subject that has been tested for a disease, disorder, or condition, and / or to whom therapy has been administered. In some instances, a human subject can be interchangeably referred to as a “patient” or “individual.” A subject administered an agent associated with treatment of a disease, disorder, or condition with which the subject is associated can be referred to as a subject in need of the agent, i.e., as a subject in need thereof.

[0130] Therapeutically effective amount: As used herein, “therapeutically effective amount” refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that a therapeutically effective amount does not necessarily achieve successful treatment in every particular treated individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0131] Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result. In some embodiments, such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and / or of a subject who exhibits only early signs of the disease, disorder, or condition. Alternatively or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, or condition. II. Multispecific Antigen Binding Constructs

[0132] The present disclosure includes a variety of binding agents that include domains provided herein, and in particular binding agents that included at least one APP-binding domain and at least one mask domain that modulates APP binding by the APP-binding domain, where the mask domain is associated with a proteolytically cleavable linker. In some embodiments, a binding agent encompassed by the present disclosure can include at last one target cell-binding domain. In some embodiments, a binding agent encompassed by the present disclosure can include two target cell- binding domains, e.g., where two target cell-binding domains target the same epitope and / or antigen, or where two target cell-binding domains target different epitopes and / or antigens.

[0133] In some embodiments, any of the multispecific antigen binding agent constructs can be a therapeutic binding agents. The multispecific antigen binding construct comprise multiple antigen binding domains to bind antigens for therapeutic uses. In embodiments herein, the multispecific construct comprises a first antigen binding domain that binds to inhibit an anti- phagocytic 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 the APP.

[0134] In some embodiments, a provided multispecific antigen binding construct comprises: (i) a first antigen binding domain that binds to and inhibits an anti-phagocytic 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 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. In some embodiments, the linker comprising the proteolytically cleavable linker joins the second antigen binding domain to the first antigen binding domain or to the immunoglobulin Fc region. In some embodiments, the first antigen binding domain and the second antigen binding domain are joined by the linker comprising the proteolyticallycleavable linker. In some embodiments, the immunoglobulin Fc region and the second antigen binding domain are joined by the linker comprising the proteolytically cleavable linker.

[0135] In some embodiments, a binding agent encompassed by the present disclosure includes an antibody structure that is linked with an APP-binding domain and a mask domain. In some embodiments, the antibody structure can have the structure of any antibody or antibody fragment provided herein. In some embodiments, the antibody structure can have a four-chain immunoglobulin structure that includes, or essentially includes, two immunoglobulin heavy chains and two immunoglobulin light chains, or an art-recognized variant thereof, e.g., as disclosed herein. In some embodiments, an antibody structure can include an immunoglobulin Fc domain. In some embodiments, an antibody structure can include an immunoglobulin Fc domain formed from constant domains of two immunoglobulin heavy chains. In some embodiments, an Fc domain can be present in an antibody structure that includes two antibody binding domains each formed by association of a heavy chain variable domain and a light chain variable domain. In some embodiments, an Fc domain can be present in an antibody structure that includes two antibody binding domains each formed by association of a heavy chain variable domain present in an immunoglobulin heavy chain and a light chain variable domain present in an immunoglobulin light chain. In some embodiments, an Fc domain can be present in an antibody structure that includes two antibody binding domains of which one or each is formed by association of heavy and light chain variable domains present in a fragment antigen-binding (Fab) domain. In some embodiments, an Fc domain can be present in an antibody structure that includes two antibody binding domains of which one or each is formed by association of heavy and light chain variable domains present in an antibody fragment such as an scFv. In some embodiments, an Fc domain can be present in an antibody structure that includes two antibody binding domains of which one or each is present in a variable domain of a heavy chain-only antibody (a VHH).

[0136] In some embodiments, a binding agent encompassed by the present disclosure includes an antibody structure that is linked at a first amino acid with an APP-binding domain, optionally via a linker, and linked at a second amino acid with a mask domain, via a proteolytically cleavable linker. In some embodiments, a binding agent encompassed by the present disclosure includes an antibody structure that is linked at a first amino acid with an APP-binding domain, optionally via a linker, the APP-binding domain is further linked at an amino acid thereof (e.g., a terminal amino acid of the APP-binding domain) , via a proteolytically cleavable linker, with a mask domain. It will be appreciated from the present disclosure that the APP-binding domain and mask domain are disposed in the binding agent such that the mask domain is able to interact with (e.g., bind) the APP-binding domain in a manner that reduces its APP-binding activity.

[0137] In some embodiments, when the linker comprising the proteolytically cleavable linker is in an uncleaved state, the second antigen-binding domain inhibits or reduces the binding of the firstantigen-binding domain to the APP. In some embodiments, when the linker comprising the proteolytically cleavable linker has been proteolytically cleaved, the second antigen binding domain does not interfere with the binding of the first antigen-binding domain to the APP.

[0138] In some embodiments, an APP-binding domain is associated with an antibody structure of a binding agent disclosed herein via an amino acid (e.g., a terminal amino acid) of an Fc domain and / or heavy chain constant domain. In some embodiments, an APP-binding domain is associated with an antibody structure of a binding agent disclosed herein via an amino acid (e.g., a terminal amino acid) of a light chain constant domain. In some embodiments, an APP-binding domain is associated with an antibody structure of a binding agent disclosed herein via an amino acid (e.g., a terminal amino acid) of light chain variable domain (e.g., of a light chain variable domain present in a Fab). In some embodiments, an APP-binding domain is associated with an antibody structure of a binding agent disclosed herein via a proteolytically cleavable linker.

[0139] In some embodiments, a target cell-binding domain is associated with an antibody structure of a binding agent disclosed herein via an amino acid (e.g., a terminal amino acid) of an Fc domain and / or heavy chain constant domain. In some embodiments, a target cell-binding -binding domain is associated with an antibody structure of a binding agent disclosed herein via an amino acid (e.g., a terminal amino acid) of a light chain constant domain. In some embodiments, a target cell- binding - binding domain is associated with an antibody structure of a binding agent disclosed herein via an amino acid (e.g., a terminal amino acid) of a light chain variable domain (e.g., of a light chain variable domain present in a Fab).

[0140] In some embodiments, a target cell-binding domain is associated with an antibody structure of a binding agent disclosed herein via a linker that does not contain a motif cleaved and / or substantially cleaved by a human protease. In some embodiments, a target cell-binding domain is associated with an antibody structure of a binding agent disclosed herein via a linker that does not contain a motif known to be cleaved by a human protease and / or known to be substantially cleaved by a human protease.

[0141] In some embodiments, a target cell-binding domain is associated with an antibody structure of a binding agent disclosed herein via a linker that does not contain a motif cleaved and / or substantially cleaved by a human protease in a microenvironment of therapeutic interest. In some embodiments, a target cell-binding domain is associated with an antibody structure of a binding agent disclosed herein via a linker that does not contain a motif known to be cleaved by a human protease, and / or known to be substantially cleaved by a human protease, in a microenvironment of therapeutic interest.

[0142] In some embodiments, a target cell-binding domain is associated with an antibody structure of a binding agent disclosed herein via a linker that does not contain a motif that is cleaved and / or substantially cleaved by a protease that cleaves and / or substantially cleaves the proteolyticallycleavable linker associated with the APP-binding domain. In some embodiments, a target cell- binding domain is associated with an antibody structure of a binding agent disclosed herein via a linker that does not contain a motif that is known to be cleaved and / or substantially cleaved by a protease that is known to cleave and / or substantially cleave the proteolytically cleavable linker associated with the APP-binding domain.

[0143] In some embodiments, a binding agent encompassed by the present disclosure is labeled in order to facilitate detection. In some embodiments, construct labeling encompasses direct labeling of the construct by coupling (i.e., physically linking) a detectable substance, as well as indirect labeling of the antibody by reactivity with a detectable substance. Labels and methods for labeling are well-known in the art and include, without limitation a radioactive agent, a radioisotope, a fluorescent compound, a fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)), a chemiluminescent compound, an enzyme, an enzyme co-factor, or any other labels known in the art. In some embodiments, enzymes that may 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 derivatives thereof (e.g., FITC); cyanine and derivatives thereof (e.g., indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine); rhodamine; Oregon green; eosin; texas red; nile red; nile blue; cresyl violet; oxazine 170; proflavin; acridine orange; acridine yellow; auramine; crystal violet; malachite green; porphin; phthalocyanine; bilirubin; allophycocyanin (APC); green fluorescent protein (GFP) and variants thereof (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 may be used as a label include biotin, avidin, and streptavidin. A. APP-Binding Domains

[0144] Binding agents encompassed by the present disclosure include a domain that binds an anti-phagocytic protein (APP; an APP-binding domain). The process of phagocytosis is naturally employed in the removal of dying or pathogenic cells. Various mechanisms limit the frequency with which healthy cells are phagocytosed, one of which is a balance of surface-expressed signal molecules that promote or activate phagocytosis (sometimes referred to as “eat-me” signals) and others that suppress phagocytosis (sometimes referred to as “don't-eat-me” signals). However, “don’t-eat-me” signals that suppress phagocytosis, when expressed by disease cells, can cause, contribute to, and / or exacerbate disease by permitting disease cells to escape phagocytosis.

[0145] Eat-me signals include antibody and complement opsonins, exposed phosphatidylserine (PS), calreticulin, oxidized low-density lipoprotein, cell-bound thrombospondin (TSP), modified intracellular adhesion molecule ICAM-3, annexin I, and other modifications to surface proteins. Don't-eat-me signaling can include anti-phagocytic receptors that mediate therecognition of don't-eat-me signals. For example, the CD47-SIRPα axis is among the most studied don't-eat-me checkpoints. In various cancers, cancer cells can overexpress CD47 and / or CD24, which enables immune evasion from macrophages.

[0146] Proteins that participate in “don’t-eat-me” signaling can be referred to as APPs. In some embodiments, an APP is expressed on myeloid cells. In some embodiments, an APP is expressed on a cell targeted for phagocytosis (e.g., a cancer cell). In some embodiments, an APP is a protein other than a receptor or ligand for a “don’t-eat-me” signaling pathway that inhibits myeloid cell phagocytosis function, (e.g., LILRB2) and such an APP may similarly be expressed on a myeloid cell or a cell targeted for phagocytosis (e.g., a cancer cell).

[0147] Without wishing to be bound by any particular scientific theory, CD47 expression on cells acts as a marker of “self” that suppresses phagocytosis by its interaction with SIRPα. CD47 (also known as integrin-associated protein, OV-3, and Rh-related protein). CD47 is a conserved, ubiquitously expressed 45–55-kDa transmembrane glycoprotein belonging to the Ig superfamily. CD47 has a single N-terminal extracellular immunoglobulin variable region (IgV) domain followed by five hydrophobic membrane-spanning segments and a short C-terminal cytoplasmic tail that is alternately spliced to form four isoforms. SIRP⍺ (also known as SIRP⍺1, PTPNS1, SHPS-1, BIT, p84, MFR, MyD-1, and CD172a) is a 115-120 kDa glycoprotein of the SIRP paired receptor family. It is expressed in most tissues and enriched, e.g., on monocytes, macrophages, CD8α classical type II dendritic cells (cDC2), neutrophils, and osteoclasts, as well as microglia and neurons.

[0148] There are other receptors and ligands, in addition to SIRPα and CD47, that can also act to suppress immune cell functions, such as that of myeloid cells. For example, CD31 (also known as PECAM-1) and inhibitory receptors of the CD300 family including CD300a and CD300f can suppress phagocytosis. CD300a and CD300f are expressed on myeloid cells and bind ligands, such as phosphatidylserine (PS).

[0149] Siglecs, or sialic acid-binding Ig-like lectins, are a large family of receptors that can suppress phagocytosis and include, e.g., CD33 (also called Siglec-3), CD22 (also called Siglec-2), and SIGLEC10. CD33 is expressed, e.g., on cells of the myeloid lineage and microglia. CD22 is expressed, e.g., in B cells, as well as myeloid-derived cells and microglia. SIGLEC10 is expressed on myeloid cells and some lymphocytes, and participates in a signaling axis with CD24.

[0150] PD-1 (also known as programmed cell death protein 1 and CD279) is a well-known immune inhibitory receptor with two cognate ligands known as PD-L1 (also known as CD274 and B7-H1; expressed, e.g., on non-lymphoid cells, and PD-L2 (also known as CD273 and B7-DC; expressed, e.g., on antigen-presenting cells). In addition to being expressed on lymphocytes, PD-1 expression can be induced in macrophages, e.g., by infection.

[0151] Human LILRB1 (leukocyte immunoglobulin-like receptor B1; also known as CD85J, ILT2, LIR-1) is an inhibitory receptor expressed on myeloid cells and subsets of lymphoid cells.LILRB1 binds 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. In particular, without wishing to be bound by any particular scientific theory, a β2 microglobulin (B2M) subunit of the MHC I complex mediates interaction between MHC I and LILRB1. MHC class I expression can protect cells from phagocytosis via engagement of LILRB1.

[0152] In some embodiments, an APP-binding domain encompassed by the present disclosure binds an APP listed in the following table: Table 1* Included in Table 1 are RNA nucleic acid molecules (e.g., thymines replaced with uredines); nucleic acid molecules encoding orthologs of the encoded proteins; DNA or RNA nucleic acid sequences comprising a nucleic acid sequence 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 more identity across their full length with any nucleic acid sequence listed in Table 1, or a portion thereof; orthologs of any protein listed in Table 1; and amino acid sequences comprising an amino acid sequence 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 more identity to across their full length with any amino acid sequence listed in Table 1, or a portion thereof. Such nucleic acid or amino acid portions can have a function of the full-length nucleic acid or amino acid molecule, respectively, as described further herein.

[0153] In some embodiments, an APP-binding domain encompassed by the present disclosure inhibits or reduces binding an APP (e.g., an 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)), with a binding partner thereof, e.g., a binding partner expressed by or present on myeloid cells, e.g., a binding partner disclosed herein.

[0154] In some embodiments, an APP-binding domain encompassed by the present disclosure inhibits or reduces binding an APP (e.g., an APP selected from PD-1, SIRPα, SIGLEC10,LILRB1, and LILRB2), with a binding partner thereof, e.g., a binding partner expressed by or present on cells targeted for modulation (e.g., direct killing and / or indirect killing) such as cancer cells, e.g., a binding partner disclosed herein.

[0155] In some embodiments, an APP-binding domain encompassed by the present disclosure binds CD47 or SIRPα. In some embodiments, the APP-binding domain inhibits the interaction between CD47 expressed on a cell targeted for modulation (e.g., direct killing and / or indirect killing) and SIRPα expressed on a myeloid cell (e.g., a macrophage, dendritic cell, monocyte, or neutrophil). In some embodiments, an APP-binding domain encompassed by the present disclosure binds CD24 or SIGLEC10. In some embodiments, the APP-binding domain inhibits the interaction between CD24 expressed on a cell targeted for modulation (e.g., direct killing and / or indirect killing) and SIGLEC10 expressed on a myeloid cell (e.g., a macrophage, dendritic cell, monocyte, or neutrophil). In some embodiments, an APP-binding domain encompassed by the present disclosure binds a PD-1 ligand, such as PD-L1 and / or PD-L2, or PD-1. In some embodiments, the APP-binding domain inhibits the interaction between the PD-1 ligand(s) expressed on a cell targeted for modulation (e.g., direct killing and / or indirect killing) and PD-1 expressed on a myeloid cell (e.g., a macrophage, dendritic cell, monocyte, or neutrophil). In some embodiments, an APP-binding domain encompassed by the present disclosure binds β2-microglobulin (B2M) or MHC-I or LILRB1. In some embodiments, the APP-binding domain inhibits the interaction between either B2M or MHC-I expressed on a cell targeted for modulation (e.g., direct killing and / or indirect killing) and LILRB1 expressed on a myeloid cell (e.g., a macrophage, dendritic cell, monocyte, or neutrophil). In other embodiments of the immediately preceding example, LILRB2 rather than LILRB1 is the myeloid cell-expressed APP. In some embodiments, the APP-binding domain binds LILRB2 expressed on a myeloid cell (e.g., a macrophage, dendritic cell, monocyte, neutrophil, tumor associated macrophage (TAM), tumor infiltrating macrophage (TIM), or a myeloid-derived suppressor cell (MDSC)) in order to block inhibition of phagocytosis by the myeloid cell.

[0156] In some embodiments, the multispecific antigen binding construct comprises an antigen-binding domain. In embodiments, multispecific antigen binding construct comprises a first antigen binding domain. In some 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 the cell surface-expressed protein; or (iii) a variant of the ECD or the binding fragment of the cell surface-expressed protein that is engineered to improve binding to the APP. In some embodiments, the binding fragment of the cell surface-expressed protein comprises a portion of the ECD of the cell surface-expressed protein. In some embodiments, the binding fragment of the cell surface-expressed protein comprises an 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 essentially of an immunoglobulin variable (V) region (domain 1) of the ECD of the cell surface-expressed protein.

[0157] In some embodiments, the multispecific antigen binding construct comprises a first antigen binding domain comprising (i) a domain of a wild-type SIRPα that binds to an anti-phagocytic protein (APP), or (ii) a variant thereof comprising one or more amino acid substitutions in the domain of the wild-type SIRPα that improves binding to the APP, wherein the APP is CD47; and a second antigen binding domain that is an anti-SIRPα antibody or antigen binding fragment that binds to the first antigen binding domain and inhibits or reduces the interaction between the first antigen binding domain and the APP; wherein the first antigen binding domain and the second antigen binding domain are joined by the proteolytically cleavable linker.

[0158] In some embodiments, an APP-binding domain binds an APP that is expressed by and / or associated with target cells of therapeutic interest. Binding agents encompassed by the present disclosure that include an APP-binding domain can cause phagocytosis of target cells.

[0159] In some embodiments, a binding agent encompassed by the present disclosure includes an APP-binding domain that binds an APP expressed by a cell or cell type that is associated with, characterized by, representative of, causes, contributes to, and / or for which phagocytosis would contribute to treatment of, a condition of interest. In some embodiments, the condition of interest is a cancer. In some embodiments, the cancer is selected from, but not limited to, adenocarcinoma, bile duct (biliary) cancer, bladder cancer, bone cancer, breast cancer (e.g., triple-negative breast cancer or Her2-negative breast cancer), carcinoid cancer, cervical cancer, cholangiocarcinoma, colon, colorectal, endometrial, esophageal cancer, glioma, head and neck cancer, (e.g., head and neck squamous cell cancer), leukemia, liver cancer, lung cancer (e.g., NSCLC, SCLC), lymphoma, melanoma, osopharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer (e.g., metastatic castration-resistant prostate carcinoma), renal cancer, sarcoma, skin cancer, squamous cell cancer, stomach cancer, testis cancer, thyroid cancer, urogenital cancer, and urothelial cancer. In some embodiments, the cancer is selected from, but 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 glioblastomas or medulloblastomas); head and / or neck cancer, breast cancers, cancers of the circulatory system (e.g., heart, mediastinum and pleura, and other intrathoracic organs, vascular cancers, and tumor-associated vascular tissue); cancers of the blood and lymphatic system (e.g., Hodgkin's disease, Non-Hodgkin's disease lymphoma, Burkitt's lymphoma, AIDS-related lymphomas, malignant immunoproliferative diseases, multiple myeloma, and malignant plasma cell neoplasms, lymphoid leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell type, leukemia of unspecified cell type, unspecified malignant neoplasms of lymphoid, haematopoietic and related tissues, such as diffuse large cell lymphoma, T- cell lymphoma or cutaneous T-cell lymphoma); cancers of the excretory system (e.g., kidney, renal pelvis, ureter, bladder, and other urinary organs); cancers of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus, and anal canal);cancers involving the liver and intrahepatic bile ducts, gall bladder, and other parts of the biliary tract, pancreas, and other digestive organs; cancers of the oral cavity (e.g., lip, tongue, gum, floor of mouth, palate, parotid gland, salivary glands, tonsil, oropharynx, nasopharynx, puriform sinus, hypopharynx, and other sites of the oral cavity); cancers of the reproductive system (e.g., vulva, vagina, cervix uteri, uterus, ovary, and other sites associated with female genital organs, placenta, penis, prostate, testis, and other sites associated with male genital organs); cancers of the respiratory tract (e.g., nasal cavity, middle ear, accessory sinuses, larynx, trachea, bronchus and lung, such as small cell lung cancer and non-small cell lung cancer); cancers of the skeletal system (e.g., bone and articular cartilage of limbs, bone articular cartilage and other sites); cancers of the skin (e.g., malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and cancers involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneoum and peritoneum, eye and adnexa, thyroid, adrenal gland, and other endocrine glands and related structures, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasms of other sites

[0160] In some embodiments, the condition is selected from, e.g., an infection (e.g., bacterial infection and / or viral infection), atherosclerosis, cardiovascular disease (e.g., heart failure following myocardial infarction), autoimmune disease (e.g., system lupus erythematosus (SLE), autoimmune nephritis, autoimmune uveitis, or autoimmune valvular carditis), organ transplant rejection, fibrotic diseases, or neurological diseases. In some embodiments, a binding agent encompassed by the present disclosure includes an APP-binding domain that binds an APP expressed by cells or cell types that are further characterized by expression of a target cell antigen encompassed by the present disclosure.

[0161] In some embodiments, an APP-binding domain can be a receptor protein. In some embodiments, an APP-binding domain can be a fragment or domain of a receptor protein. In some embodiments, an APP-binding domain can be a receptor ligand. In some embodiments, an APP- binding domain can be a fragment or domain of a receptor ligand. In some embodiments, an APP- binding domain can be an antibody or antibody fragment. In some embodiments, an APP-binding domain can be a small molecule. In some embodiments, an APP-binding domain can be an aptamer.

[0162] In some embodiments, the APP-binding domain is a SIRPα protein. In some embodiments, the APP-binding domain is a fragment or domain of a 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 a 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 a PD-1 protein. In some embodiments, the APP-binding domain is an LILRB1 protein. In some embodiments, the APP-binding domain is a fragment or domain of an LILRB1 protein. In someembodiments, the APP-binding domain is an LILRB2 protein. In some embodiments, the APP- binding domain is a fragment or domain of an 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 a 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 a PD-L2 protein. In some embodiments, the APP-binding domain is a CD47 protein. In some embodiments, the APP-binding domain is a fragment or domain of a CD47 protein. In some embodiments, the APP-binding domain is a CD24 protein. In some embodiments, the APP-binding domain is a fragment or domain of a CD24 protein. In some embodiments, the APP-binding domain is a β2M protein. In some embodiments, the APP-binding domain is a fragment or domain of a β2M protein. In some embodiments, the APP-binding domain is a protein of an 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 an MHC-1 complex (e.g., HLA-A, HLA-B, or HLA-C).

[0163] In some embodiments, an APP-binding domain can comprise an antibody or an antigen-binding portion thereof. In some embodiments, the APP-binding antibody or antigen-binding portion thereof can include at least one immunoglobulin heavy chain and / or at least one immunoglobulin light chain. In some embodiments, an APP-binding antibody or antigen-binding portion thereof can include at least one immunoglobulin heavy chain variable domain and / or at least one immunoglobulin light chain variable domain. In some embodiments, an APP-binding antibody or antigen-binding portion thereof can include CDR1, CDR2, and CDR3 of at least one immunoglobulin heavy chain variable domain and / or CDR1, CDR2, and CDR3 of at least one immunoglobulin light chain variable domain. In some embodiments, APP-binding antibodies or antigen-binding portions thereof can be or include an extracellular domain (ECD) of a cell surface-expressed protein, a mutated version (variant) of the ECD of the cell surface-expressed protein that is engineered to improve binding to target, intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti- idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies® minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies,, Adnectins®, Affilins®, Trans- bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, KALBITOR®s, CARs, engineered TCRs, and antigen-binding fragments of any of the foregoing.

[0164] In some embodiments, the APP-binding domain is a variant APP-binding domain comprising one or more amino acid substitutions that improves binding to CD47. In some embodiments, the binding of a variant APP-binding domain comprising one or more amino acidsubstitutions in the IgV domain to CD47 is improved compared to the binding of the wild-type APP- IgV domain. In some embodiments, the APP- binding domain binds to CD47 with a dissociation constant (KD) of less than 100 picomolar (pM). In some examples, the variant APP- binding domain binds to CD47 with a dissociation constant (KD) of less than 100 picomolar (pM).

[0165] In some embodiments, the affinity (KD) of an APP-binding domain encompassed by the present disclosure for a target APP may be about 0.002 to about 200 nM. In some embodiments, the binding affinity of an APP-binding domain encompassed by the present disclosure for a target APP may be any of about 250 nM, 200 nM, about 100 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 2 pM, or less. In some embodiments, the binding affinity is less than any of 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 less, or any range in between, such as about 5 nM to about 35 nM. In some embodiments, the binding affinity of an APP-binding domain encompassed by the present disclosure for a target APP may be approximately less than 1 x 10-7M, such as approximately less than 10-8M, 10-9M, 10-10M, 10-11M, or lower. In some embodiments, affinity (KD) can be determined using well-known assays in the art, such as by using surface plasmon resonance (SPR) technology in a BIACORE® assay instrument. 1. CD47 APP Binding Domains

[0166] 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 modulators of this pathway generally target the ubiquitously expressed CD47 cell surface molecule (see, e.g., Chao et al. Cell 2010 Sep 3;142(5):699-713; Weiskopf et al. Science.2013 Jul 5;341 (6141 ):88-91 ). As described herein, CD47 APP binding domains can be used inhibit or block to interaction of CD47 and endogenous or wild-type SIRPα. For example, CD47 APP binding domains that bind CD47 can enable phagocytosis of solid tumor cells, inhibit tumor growth and prevent metastasis tumor cells. Provided herein are multispecific antigen binding constructs that contain CD47 APP binding domains. These multispecific constructs have specificity for CD47 via the CD47 binding domain and also can comprise a binding domain with specificity for another antigen. In some embodiments, the multispecific antigen binding construct contains a CD47 APP binding domain comprising a SIRPα polypeptide or a variant or fragment thereof. In other embodiments, the multispecific antigen binding construct contains a CD47 APP binding domain that is not a SIRPα polypeptide. In theseembodiments, the multispecific antigen binding constructs can comprise a CD47 APP binding domain that is an anti-CD47 antibody, or antigen binding fragment thereof. In embodiments of a multispecific antigen binding construct provided herein, a first antigen binding domain is a CD47 binding domain, such as SIRPα. (i) SIRPα

[0167] The SIRPα protein is 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 cytoplasmic region. The SIRPα extracellular domain (ECD) contains a single N-terminal IgV-like domain (IgV domain), followed by two IgC-like domains, a transmembrane domain, and a cytoplasmic tail. The IgV domain (D1) of SIRPα can interact with CD47. The interaction of the N-terminal IgV domain of CD47 with the SIRPα IgV / D1 to promote phosphorylation of tyrosine residues. An exemplary full length SIRPα protein is set forth a Uniprot Accession No. P78324.

[0168] CD47 functions as a ligand for SIRPα. Upon binding of endogenous SIRPα on the macrophage cell surface to CD47, SIRPα initiates signal to inhibit phagocytosis of the CD47 containing cell. The binding interface between SIRPα and CD47, as well as residues of both proteins that participate in binding, are known and previously described in the art (see e.g., Hatherley et al. (2007) J. Biol. Chem.282:14567-75; Nakaishi et al. (2008) J. Mol. Biol.375:650-60). As shown herein, binding of a multispecific antigen binding construct comprising a SIRPα polypeptide, such as a variant SIRPα polypeptide (e.g., variant SIRPα polypeptide with increased affinity for CD47 compared to wild-type SIRPα) can be used for disrupting binding of endogenous SIRPα to CD47 on the cell surface. A SIRPα polypeptide, such as SIRPα polypeptides provided herein, can bind CD47 and inhibit the interaction of CD47 and endogenous or wild-type SIRPα.

[0169] In some embodiments, the multispecific antigen binding construct comprises an antigen binding protein that binds an anti-phagocytic protein (APP) that is CD47, such as an antigen binding protein that is SIRPα. In embodiments herein, the antigen binding protein is a SIRPα that binds an anti-phagocytic protein (APP) that is CD47. In some embodiments, the SIRPα polypeptide binds CD47 and inhibits the interaction of CD47 and SIRPα. In some embodiments, the SIRPα binds CD47 on diseased cells (e.g., tumor cells) with higher affinity compared to CD47 on non-diseased cells.

[0170] In some embodiments, a SIRPα in a multispecific antigen binding construct provided herein inhibits binding between an extracellular domain or binding fragment thereof of a wild-type SIRPα polypeptide and CD47. In some embodiments, a SIRPα provided herein inhibits binding between of a wild-type SIRPα and an IgSF domain of a CD47 protein. In embodiments herein, the CD47 is a human CD47 protein.

[0171] In embodiments, the SIRPα in a multispecific antigen binding construct comprises a SIRPα polypeptide selected from (i) an ECD of the wild-type SIRPα; (ii) a binding fragment of the wild-type SIRPα; and (iii) a variant of the ECD or the binding fragment of the wild-type SIRPα that is engineered to improve binding to the CD47.

[0172] In some embodiments, the multispecific antigen binding construct comprises a wild- type SIRPα polypeptide or portion thereof. In some embodiments, the SIRPα in a multispecific antigen binding construct comprises a domain of wild-type SIRPα.In some embodiments, the SIRPα in a 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 consisting essentially of extracellular domain of SIRPα. In some embodiments the SIRPα is a binding fragment of wild-type SIRPα that binds CD47. In some embodiments the SIRPα is a binding fragment of the wild-type SIRPα ECD and that binds CD47. In some embodiments, a binding fragment of SIRPα comprises an immunoglobulin variable (V) region (IgV; also termed D1) of the ECD of the SIRPα. In some embodiments, the SIRPα polypeptide is binding fragment of a wild-type SIRPα polypeptide that comprises the SIRPα IgV domain and that binds CD47. In any of the preceding embodiments, the SIRPα polypeptide is an APP-binding domain.

[0173] In some embodiments, the multispecific antigen binding construct comprises a variant SIRPα polypeptide. A variant SIRPα polypeptide can comprise 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, a variant SIRPα polypeptide provided herein comprises 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 the extracellular domain, such as wild-type SIRPα polypeptide described herein. The one or more amino acid modifications, e.g. substitution, can be in the ectodomain (extracellular domain) of the reference (e.g., unmodified or wild-type) SIRPα sequence. In some embodiments, the one or more amino acid modifications is in D1 of SIRPα.

[0174] Unless stated otherwise, as indicated throughout the present disclosure, the amino acid modification(s) in a variant SIRPα polypeptide are designated by amino acid position number corresponding to the numbering of positions of the sequence of the unmodified or wild type SIRPα ECD sequence set forth in SEQ ID NO:206, or a portion thereof set forth in SEQ ID NO.103 or 104. It is within the level of a skilled artisan to identify the corresponding position of a modification, e.g. amino acid substitution, in a SIRPα polypeptide, including portion thereof containing the IgV domain thereof, such as by alignment of a variant SIRPα sequence with the sequence of amino acids set forth in SEQ ID NO:103 or 104. In reference to amino acid substitutions throughout this disclosure, 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 is listed afterthe number. For example, the amino acid substitution N80A refers to a replacement of asparagine (N) at position 80 with alanine (A), where position 80 is the 80thamino acid in the sequence of amino acids set forth in SEQ ID NO.103 or 104.

[0175] In some embodiments, the SIRPα into which the amino acid modifications are introduced, is a wild-type SIRPα comprising the extracellular domain of SIRPα, such as any wild-type SIRPα described herein. In an exemplary embodiment, the variant SIRPα comprises one or more amino acid modifications in a wild-type SIRPα comprising the extracellular domain of SIRPα. However, the variant SIRPα polypeptide need not comprise the entire extracellular domain (ECD). In some embodiments, the variant SIRPα comprises one or more amino acid modifications in a wild-type SIRPα comprising a portion of the ECD of SIRPα, such as a binding fragment of SIRPα that binds CD47. In some embodiments, the one or more amino acid modification, e.g. substitution is in the SIRPα ECD or portion thereof containing the IgV. In some embodiments, the variant SIRPα comprises one or more amino acid modifications in a wild-type SIRPα comprising the IgV domain of SIRPα. In some embodiments, the variant SIRPα comprises one or more amino acid modifications binding fragment of the SIRPα ECD comprising D1. In some embodiments, the variant SIRPα comprises one or more amino acid modifications in te sequence of amino acids set forth in SEQ ID NO.103 or 104. In some embodiments, the variant SIRPα comprises one or more amino acid modifications in the sequence of amino acids set forth in SEQ ID NO.103. In some embodiments, the variant SIRPα comprises one or more amino acid modifications in the sequence of amino acids set forth in SEQ ID NO.103.

[0176] In some embodiments, the variant SIRPα polypeptide is a soluble polypeptide and lacks a transmembrane domain.

[0177] In embodiments herein, a variant SIRPα polypeptide(s) exhibits altered (e.g., increased) binding affinity for CD47. In some embodiments, the variant SIRPα binds CD47 with higher affinity than wild type SIRPα. In embodiments herein, a variant SIRPα comprises one or more amino acid substitutions in a wild-type SIRPα that alters binding to CD47. In some embodiments, a variant SIRPα comprises one or more amino acid substitutions in a domain of a wild-type SIRPα that improves binding to CD47. In particular embodiments, a variant SIRPα comprises one or more amino acid substitutions in the extracellular domain of a wild-type SIRPα or binding fragment of the SIRPα ECD, that improves binding to CD47 compared to binding of a wild-type SIRPα not containing the one or more amino acid substitutions. In embodiments herein, the variant SIRPα comprises a binding fragment of the wild-type SIRPα that is engineered to improve binding to the CD47. In embodiments, a binding fragment of SIRPα comprises D1.In embodiments herein, the variant SIRPα comprises the extracellular domain of the wild-type SIRPα that is engineered to improve binding to the CD47. In embodiments herein, the variant SIRPα consists essentially of the extracellular domain of the wild- type SIRPα that is engineered to improve binding to the CD47. Improved binding of a variant SIRPαcompared to a wild-type or unmodified SIRPα can be demonstrated by a variant SIRPα exhibiting higher affinity for CD47 compared to wild type SIRPα. Higher affinity of a variant SIRPα can be demonstrated by a smaller the dissociation constant (KD) compared to a wild-type SIRPα. Thus, the smaller the dissociation constant the more tightly bound the ligand (e.g., CD47) is, or the higher the affinity between ligand and protein (e.g., between CD47 and SIRPα). In some embodiments, the variant SIRPα binds CD47 on diseased cells (e.g., tumor cells) with higher affinity compared to CD47 on non-diseased cells.

[0178] In some embodiments, a SIRPα as provided herein, such as, for example, a variant SIRPα, binds to CD47 wherein binding of wild-type or endogenous SIRPα to CD47 is blocked. For example, the variant SIRPα and the wild type SIRPα (e.g., endogenous) polypeptide may "compete" for the same CD47 epitope. In some examples, a variant SIRPα or wild type SIRPα in a multispecific antigen binding construct can outcompete wild-type or endogenous SIRPα for binding to CD47. In some embodiments where a variant SIRPα outcompetes a wild type (e.g., endogenous) a SIRPα for binding to CD47, the variant SIRPα has increased affinity for CD47 compared to the wild type (e.g., endogenous) SIRPα.

[0179] In embodiments where the SIRPα polypeptide comprises a SIRPα binding fragment, the fragment or portion of the SIRPα polypeptide is sufficient to bind CD47. In some of any embodiments, the specific binding fragment is less than the full-length ECD set forth in SEQ ID NO:206. In some embodiments, a SIRPα binding fragment (also SIRPα binding region herein) comprises an immunoglobulin variable (V) region (domain 1) of the ECD of SIRPα. In some embodiments, the SIRPα binding fragment contains the sequence set forth in SEQ ID NO.103. In some embodiments, the SIRPα binding fragment contains the sequence set forth in SEQ ID NO.104. In some embodiments, a SIRPα binding fragment consists essentially of an immunoglobulin variable (V) region (domain 1) of the ECD of SIRPα. In some embodiments, the SIRPα binding fragment consists essentially of the sequence set forth in SEQ ID NO.103. In some embodiments, the SIRPα binding fragment contains consists essentially of the sequence set forth 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.

[0180] In some embodiments, the SIRPα is a SIRPα binding region that is 100 to 120 amino acids in length, such as 105 to 115, 105 to 110, 105 to 115, 110 to 120, 110 to 115, 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 embodiments herein, the SIRPα binding region is at or 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 examples the SIRPα binding region is at or about 115 amino acids in length.

[0181] In some embodiments, the wild-type SIRPα is a wild-type human SIRPα. In some embodiments, the variant SIRPα comprises one or more amino acid modifications, such as, for example, one or more amino acid substitutions in a wild-type human SIRPα. In embodiments herein, a wild-type human SIRPα is any allele of human SIRPα (see e.g., Takenaka Nat Immunol. 2007;8(12):1313–1323; GenBank accession no. NM001040022.1 and D86043.1). In embodiments herein, the IgV domain of wild type human SIRPα is set forth in SEQ ID NO.103 and SEQ ID NO. 104. In embodiments herein, the ECD of wild type human SIRPα is set forth in SEQ ID NO.206.

[0182] 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 modification(s), e.g. substitution, 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 or specific binding fragment thereof of the wild-type TACI sequence. The modification, e.g. substitution can be 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.

[0183] In some embodiments, the SIRPα comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the wild type SIRPα polypeptide. In some embodiments, the SIRPα comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the wild type SIRPα polypeptide wherein the wild-type SIRPα polypeptide comprises (i) the sequence of amino acids set forth in SEQ ID NO: 206 or (ii) a portion of the SIRPα ECD comprising an IgV domain of SIRPα. In particular embodiments, the SIRPα comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the wild type SIRPα polypeptide wherein the wild-type SIRPα polypeptide comprises the IgV domain of SIRPα that has 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.

[0184] In some embodiments, the SIRPα comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:104. In some embodiments, the SIRPα is set forth by an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:104.

[0185] In some embodiments, the SIRPα comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:103. In some embodiments, the SIRPα is set forth by an amino acidsequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 103.

[0186] In some embodiments, a variant SIRPα comprises comprising one or more amino acid modifications (e.g. amino acid substations) as described has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the SIRPα polypeptide set forth in SEQ ID NO: 103 or specific binding fragment thereof. domain.

[0187] In some embodiments, a variant SIRPα comprises comprising one or more amino acid modifications (e.g. amino acid substations) as described has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the SIRPα polypeptide set forth in SEQ ID NO: 104 or specific binding fragment thereof.

[0188] In some embodiments, the SIRPα is a variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47. In some embodiments, the binding of a variant SIRPα comprising one or more amino acid substitutions in the IgV domain to CD47 is improved compared to the binding of the wild-type SIRPα IgV domain.

[0189] In some embodiments, the SIRPα binding domain binds to CD47 with a dissociation constant (KD) of with a dissociation constant (KD) of less than 100 nanomolar (nM), less than 10 nM, less than 1 nM, less than 100 picomolar (pM), less than 10 pM or less than 1 pM, or any combination between any of the foregoing. The particular SIRPα binding domain can be chosen based on the desired binding affinity. This provides a platform that allows interactions to be flexibility chosen, such as depending on the particular mask domain to be used. In some embodiments, the binding affinity of a SIRPα binding domain, such as a variant SIRPα binding domain, for CD47 is any of about 250 nM, 200 nM, about 100 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 2 pM, or less. In some embodiments, the binding affinity is less than any of 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 less, or any range in between, such as about 5 nM to about 35 nM. In some embodiments, the binding affinity of a variant SIRPα for CD47 is at or about less than 1 x 10-7M, such as approximately less than 10-8M, 10-9M, 10-10M, 10-11M, or lower. In some embodiments, affinity (KD) can be determined using well- known assays in the art, such as by using surface plasmon resonance (SPR) technology in a BIACORE® assay instrument.

[0190] For instance, in some embodiments, the SIRPα binding domain is a wild-type SIRPα binding domain or is a variant thereof in which binding to a wildtype human CD47, such as cell surface-expressed CD47, is with a dissociation constant (KD) of less than 100 nanomolar (nM). In some embodiments, the KD is from 1 nM to 100 nM, 1 nM to 75 nM, 1 nM to 50 nM, 1 nM to 25 nM, 1 nM to 10 nM, 10 nM to 100 nM, 10 nM to 75 nM, 10 nM to 50 nM, 10 nM to 25 nM, 25 nM to 100 nM, 25 nM to 75 nM, 25 nM to 50 nM, or 50 nM to 100 nM, 50 nM to 75 nM, or 75 nM to 100 nM.

[0191] In some embodiments, the SIRPα binding domain is a variant SIRPα comprising one or more amino acid substitutions in a domain (e.g., the IgV domain) of the wild-type SIRPα that improves binding to CD47. In some embodiments, the variant SIRPα has an intermediate affinity for binding to CD47. In some embodiments, the variant SIRPα binds to a wildtype human CD47, such as cell surface-expressed SIRPα, with a dissociation constant (KD) of less than 1 nM. In some embodiments, the KD is from 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 such a variant SIRPα is a SIRPα containing the mutation E54Q, in some cases as the only mutation relative to a domain of a wild-type SIRPα.

[0192] In some embodiments, the SIRPα binding domain is a variant SIRPα comprising one or more amino acid substitutions in a domain (e.g., the IgV domain) of the wild-type SIRPα that improves 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 binds to a wildtype human CD47, such as cell surface-expressed SIRPα, with a dissociation constant (KD) of less than 100 picomolar (pM). In some embodiments, the KD is from 1 pM to 100 pM, optionally from 1 pM to 75 pM, 1 pM to 50 pM, 1 pM to 25 pM, 1 pM to 10 pM, 10 pM to 100 pM, 10 pM to 75 pM, 10 pM to 50 pM, 10 pM to 25 pM, 25 pM to 100 pM, 25 pM to 75 pM, 25 pM to 50 pM, or 50 pM to 100 pM, 50 pM to 75 pM, or 75 pM to 100 pM. An example of such a variant is the variant SIRPα known as CV1. In some embodiments, the variant SIRPα has the amino acid substitutions V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I, corresponding to amino acid numbering of SEQ ID NO:103 or SEQ ID NO:104.

[0193] In some embodiments, the SIRPα binding domain, such as a variant SIRPα binding domain, binds to CD47 with a dissociation constant (KD) of from at or about 1 pM to 100pM, optionally from at or about 10 pM to 50 pM.

[0194] A variant SIRPα polypeptide provided herein can comprise one or more amino acid modifications that are known in the art to alter (e.g., improve) binding to CD47. Exemplary amino acid modifications (e.g., substitutions) are described in Lee et al., J Immunol.2007 Dec 1;179(11):7741-50; Weiskopf et al., Science.2013 Jul 5; 341(6141): 10.1126 / science.1238856; International Patent Publication No. WO 2016 / 023040). In some embodiments, a variant SIRPα comprises amino acid substitutions in the contact residues with CD47. In embodiments, a variantSIRPα comprises amino acid substitutions in the contact residues at one or more of positions A29, L30, I31 , P32, V33, G34, P35, Q52, K53, E54, S66, T67, K68, R69, F74, K93, K96, G97, S98, and D100. In some embodiments, a variant SIRPα comprises amino acid substitutions in the hydrophobic core. In embodiments, a variant SIRPα comprises amino acid substitutions in the hydrophobic core at one or more of positions L4, V6, V27, I36, F39, L48, I49, Y50, F57, V60, M72, F74, I76, V92, F94 and F103. In some embodiments, a variant SIRPα comprises a combination of amino acid substitutions selected from among 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, I31 T, 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, I31 T, 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, I31 T, E47V, K53R, E54Q, H56P, S66G, K68R, V92I, and F103V; V6I, V27I, 131 F, E47V, K53R, E54Q, H56P, S66T, and V92I.

[0195] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in a wild-type SIRPα polypeptide or specific binding fragment thereof selected from L4F or L4I or L4V, V6F or V6I or V6L, V27F or V27I or V27L, I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L or H56R, S66G or S66T or S66A, K68R, N80A, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V, or a conservative amino acid substitution thereof, corresponding to amino acid numbering of SEQ ID NO. 103. In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in a wild-type SIRPα polypeptide or specific binding fragment thereof selected from L4F or L4I or L4V, V6F or V6I or V6L, A27F or A27I or A27L, I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L or H56R, L66G or L66T or L66A, K68R, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V, or a conservative amino acid substitution thereof, corresponding to amino acid numbering of SEQ ID NO. 104.

[0196] A conservative amino acid modification, e.g. substitution is any amino acid that falls in the same class of amino acids as the substituted amino acids, other than the reference (e.g., unmodified) or wild-type amino acid. The classes of amino acids are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartate, glutamate, asparagine, and glutamine).

[0197] In some embodiments, the variant SIRPα polypeptide has one or more amino acid substitutions in a wild-type SIRPα polypeptide or specific binding fragment wherein the one more amino acid substitutions are K53R, E54Q and S66T, corresponding to amino acid numbering of SEQ ID NO: 103.

[0198] In some embodiments, the variant SIRPα polypeptide has one or more amino acid substitutions in a wild-type SIRPα polypeptide or specific binding fragment wherein the one more amino acid substitutions are K53R, E54Q and L66T, corresponding to amino acid numbering of SEQ ID NO: 104.

[0199] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in a wild-type SIRPα polypeptide or 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 amino acid numbering of SEQ ID NO:103.

[0200] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in a wild-type SIRPα polypeptide or 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 amino acid numbering of SEQ ID NO:104.

[0201] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in a wild-type SIRPα polypeptide or specific binding wherein the one or more substitutions are V6I, V27I, I31F, E47V, K53R, E54Q, H56P, S66T, and V92I, corresponding to amino acid numbering of SEQ ID NO:103.

[0202] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in a wild-type SIRPα polypeptide or specific binding wherein the one or more substitutions are V6I, A27I, I31F, E47V, K53R, E54Q, H56P, L66T, and V92I, corresponding to amino acid numbering of SEQ ID NO:104.

[0203] In some embodiments, the variant SIRPα polypeptide comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:105.

[0204] In some embodiments, the variant SIRPα polypeptide is set forth by an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:105.

[0205] In some embodiments, the variant SIRPα polypeptide comprises an amino acid sequence set forth in SEQ ID NO:105.

[0206] In some embodiments, the variant SIRPα polypeptide is set forth by an amino acid sequence set forth in SEQ ID NO:105.

[0207] In some embodiments, the SIRPα polypeptide is deglycosylated.

[0208] In some embodiments, SIRPα contains an N-glycosylation site N80 mutated to alanine (A) in the D1 region. In some embodiments the N-glycosylation site N80 is not mutated, and the glycosylation site is retained.

[0209] In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in a wild-type SIRPα polypeptide or specific binding wherein the one or more substitutions comprise N80A, corresponding to amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104. In some embodiments, the variant SIRPα polypeptide comprises one or more amino acid substitutions in a wild-type SIRPα polypeptide or specific binding wherein the one or more substitutions is N80A, corresponding to amino acid numbering of SEQ ID NO: 103 or SEQ ID NO: 104.

[0210] In some embodiments, the variant SIRPα polypeptide comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10.

[0211] In some embodiments, the variant SIRPα polypeptide is set forth by an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10.

[0212] In some embodiments, the variant SIRPα polypeptide comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:27.

[0213] In some embodiments, the variant SIRPα polypeptide is set forth by an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:27.

[0214] In some embodiments, the variant SIRPα polypeptide comprises an amino acid sequence set forth in SEQ ID NO:27.

[0215] In some embodiments, the variant SIRPα polypeptide is set forth by an amino acid sequence set forth in SEQ ID NO:27. (ii) Anti-CD47 Antibody

[0216] Blocking CD47 activity by anti CD47 antibodies was previously shown to activate macrophage phagocytosis of CD47+ cells, such as tumor cells. Administration of anti-CD47 monoclonal antibodies can decrease the tumor burden in mouse models of hematologic neoplasms and solid tumors. In embodiments herein, the multispecific antigen binding constructs can contain an anti- CD47 antibody or antigen binding fragment thereof. For example, anti-CD47 antibodies have previously been developed and / or that are known in the art can be included in the multispecificantigen binding constructs provided herein. In some embodiments, an anti-CD47 antibody of the present disclosure blocks binding between an extracellular domain (e.g., the ECD comprising the D1 domain) of a SIRP-α polypeptide and an IgSF domain of a human CD47 polypeptide. For example, the anti-CD47 antibody and the SIRP-α polypeptide may "compete" for the same CD47 epitope, and / or antibody binding to CD47 may be mutually exclusive with CD47 binding to SIRP-α.

[0217] Any of a large number of publicly available and / or known anti-CD47 can be included in the constructs described herein. Exemplary anti-CD47 antibodies or antigen binding fragments thereof include, but are not limited to, Hu5F9-G4 (also termed Magrolimab and 5F9 by Gilead Sciences, Inc.) which is 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 (from Celegene, see Clinical Trials.gov identifiers NCT02641002 and NCT02367196, and Narla. Abstract 4694, Immunology.20174694-4694); AO-176, a humanized IgF2 anti-CD37 mAb (from Arch Oncology; see Clinical Trials.gov identifier NCT04445701; Puro. Mol. Cancer Ther.2020;19(3):835–846) and the related Vx1000R mouse anti-human CD47 antibody (see Kaur. Antibody Ther.2020;3(3):179–192); SRF231 a human IgG4 anti-CD47 mAb (from Surface Oncology, see Clinical Trials.gov identifier NCT03512340; Holland. Blood.2016;128(22) 1843-1843); IMC-002, a fully human IgG4 anti-CD47 mAb (from ImmuneOncia Therapeutics; see Clinical Trials.gov identifier NCT04306224; Yoo, J. Immunother. Cancer.2020;8(Suppl 3) A237- A237); Letaplimab, a humanized anti-CD47 antibody (from Innovent Biologics; see Clinical Trials.gov identifier NCT0376149); SHR-1603 (by Jiangsu Hengrui Medicine, Co., Ltd.); TJC4 (from I-Mab Biopharma, Co., Ltd.); IBI188 (from Innovent Biologics, Inc.); and AO-176 (from Arch Oncology, Inc); or any variants or combinations thereof. In other examples, anti-CD47 monoclonal antibodies that were previously shown to block the CD47-SIRP interaction can be included in the multispecific antigen binding constructs provided herein include B6H12.2 and BRIC126 (see e.g., Subramanian et al., Blood.2006; 107:2548–2556). In other examples, the multispecific antigen binding constructs provided herein can contain Hu5F9-G4, a humanized IgG4 monoclonal antibody, that was shown to block the CD47-SIRPα interaction (Clinical Trials.gov identifier NCT02953509).Anti-CD47 Antibody or Antibody Fragment

[0218] In some embodiments, the multispecific antigen binding constructs provided herein contain an anti-CD47 single-domain antibody fragment, such as those derived from camelid heavy chain antibodies (nanobodies, VHH domains). In embodiments, VHH domains that have previously been developed can be included in the multispecific antigen binding constructs provided herein. For example, HuNb1, which is a VHH with high affinity for CD47.

[0219] In embodiments herein, the In embodiments herein, the multispecific antigen binding constructs can contain an anti-CD47 antibody or antigen binding fragment thereof that is pan reactive. For example, a multispecific antigen binding construct provided here contains an anti-CD47 antibodyor antigen binding fragment thereof that has affinity for wild-type CD47 and also has affinity for other CD47 variants. B. Mask Domains

[0220] Binding agents encompassed by the present disclosure can include a mask domain that modulates binding of an APP by the APP-binding domain. In some embodiments, the mask domain binds the APP-binding domain in a manner that, when bound, inhibits its binding of APP. The present disclosure further includes that the mask domain can be associated with a proteolytically cleavable linker, such that the mask domain inhibits binding of APP when associated with the binding agent (i.e., when the linker has not been cleaved), but inhibition of APP binding is relieved when the linker is cleaved, thereby achieving activatable activity. Accordingly, a binding agent encompassed by the present disclosure that includes an APP-binding domain and a mask domain that binds the APP binding domain, modulation of myeloid cell activity can be conditional upon cleavage of a proteolytically cleavable linker. Moreover, where one or more proteases capable of cleaving the proteolytically cleavable linker is more active or more highly expressed in a particular microenvironment (e.g., a disease microenvironment such as a cancer microenvironment), activity of the binding agent can be specific to the particular microenvironment.

[0221] In embodiments of a multispecific antigen binding construct provided herein, a second antigen binding domain is a mask domain that modulates binding of an APP by the APP- binding domain. In some embodiments of a multispecific antigen binding construct provided herein, a second antigen binding domain is a mask domain that modulates binding of CD47 to SIRPα.

[0222] In some embodiments, a first exemplary state of the binding agent can be a state in which the proteolytically cleavable linker is intact such that an associated mask domain can inhibit APP binding by an APP-binding domain. In some embodiments, a second exemplary state of the binding agent can be a state in which the proteolytically cleavable linker has been cleaved, releasing the mask domain from the binding agent. In some embodiments, the first exemplary state is characterized in that the binding agent does not, or does not significantly, induce myeloid cell activity on target cells (e.g., phagocytosis of target cells). In some embodiments, the second exemplary state is characterized in that the binding agent can induce myeloid cell activity on target cells (e.g., phagocytosis of target cells).

[0223] In some embodiments, the first exemplary state and the second exemplary state demonstrate differential effects on myeloid cells and / or target cells (e.g., direct and / or indirect target cell killing, e.g., target cell phagocytosis) between an activating condition (e.g., an activating microenvironment) and a non-activating condition (e.g., a non-activating microenvironment). In some embodiments, an activating condition refers to an environment that includes a protease capable of cleaving the proteolytically cleavable linker of a binding agent disclosed herein. In some embodiments, a non-activating condition refers to an environment that does not include a proteasecapable of cleaving the proteolytically cleavable linker of a binding agent disclosed herein. In some embodiments, a binding agent does not cause an increase, or does not cause a significant increase, in myeloid cell activity and / or target cell killing (e.g., direct and / or indirect target cell killing, e.g., target cell phagocytosis) in a non-activating condition.

[0224] In some embodiments, a binding agent causes an increase (e.g., a significant increase) in myeloid cell activity and / or target cell killing (e.g., direct killing and / or indirect killing) in an activating condition. In some embodiments, a binding agent causes an increase in or level of myeloid cell activity and / or target cell killing (e.g., direct killing and / or indirect killing) in an activating condition that is at least 10% greater than an increase in or level of myeloid cell activity and / or target cell killing (e.g., direct killing and / or indirect killing) in a non-activating condition (e.g., at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 10-fold greater, at least 100-fold greater, at least 1,000-fold greater, at least 10,000-fold greater, at least 50,000-fold greater, at least 100,000-fold greater, at least 500,000-fold greater, at least 1,000,000-fold greater, or more).

[0225] In some embodiments, the affinity of an APP-binding domain that is not bound with a mask domain (e.g., in an activating condition) for its target APP is at least 20% greater than the affinity of the APP-binding domain bound with a mask domain (e.g., in a non-activating condition). In some embodiments, the affinity of an APP-binding domain that is not bound with a mask domain (e.g., in an activating condition) for its target APP is at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 10-fold greater, at least 100- fold greater, or at least 1000-fold greater than the affinity of the APP-binding domain bound with a mask domain (e.g., in a non-activating condition).

[0226] In some embodiments, the affinity of an APP-binding domain that is not bound with a mask domain (e.g., in an activating condition) for its target APP can be equal to or less than about 200 nM. In some embodiments, the affinity of an APP-binding domain that is not bound with a mask domain (e.g., in an activating condition) for its target APP can 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, about 25 nM, about 20 nM, about 15 nM, about 10 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 2 pM, or less. In some embodiments, the binding affinity is less than any of 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 less, or any range in between, such as about 5 nM to about 35 nM. In some embodiments, the affinity of an APP-binding domain that is not bound with a mask domain (e.g., in an activating condition) for its target APP can be equal to or less than about 1 x 10-7M, such as equal to or less than about 10-8M, 10-9M, 10-10M, 10-11M, or lower. In some embodiments, affinity (KD) can be determined using well-known assays in the art, such as by using surface plasmon resonance (SPR) technology in a BIACORE® assay instrument.

[0227] In some embodiments, the affinity (KD) of an APP-binding domain that is bound with a mask domain (e.g., in a non-activating condition) can be equal to or greater than about 200 nM. In some embodiments, the affinity of an APP-binding domain that is not bound with a mask domain (e.g., in an activating condition) for its target APP can be equal to or greater than 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, 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 greater. In some embodiments, the binding affinity is greater than any of 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 greater, or any range in between, such as about 5 nM to about 35 nM. In some embodiments, the affinity of an APP-binding domain that is not bound with a mask domain (e.g., in an activating condition) for its target APP can be equal to or greater than about 1 x 10-7M, such as equal to or greater than about 10-8M, 10-9M, 10-10M, 10-11M, or lower. In some embodiments, affinity (KD) can be determined using well-known assays in the art, such as by using surface plasmon resonance (SPR) technology in a BIACORE® assay instrument.

[0228] In some embodiments, an APP-binding domain has equal to or greater than about 1- fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, about 5000-fold, or greater, or any range in between, such as about 500- fold to about 1000-fold decreased affinity for its corresponding APP when the APP-binding domain is bound by the mask domain as compared to when the APP-binding domain is not bound by the mask domain.

[0229] In some embodiments, a mask domain has a dissociation constant for binding to the APP-binding domain that is greater than the dissociation constant of the APP-binding domain forbinding to the corresponding APP. In some embodiments, a mask domain does not interfere or compete with an APP-binding domain for binding to its APP when the masking domain is cleaved from the binding agent by cleavage of a proteolytically cleavable linker.

[0230] In some embodiments, a mask domain of a binding agent encompassed by the present disclosure can be an antibody or antibody fragment that binds an APP-binding domain of the binding agent. In some embodiments, a mask domain of a binding agent encompassed by the present disclosure can be a receptor protein that binds an APP-binding domain of the binding agent. In some embodiments, a mask domain of a binding agent encompassed by the present disclosure can be a fragment or domain of a receptor protein that binds an APP-binding domain of the binding agent. In some embodiments, a mask domain of a binding agent encompassed by the present disclosure can be a receptor ligand that binds an APP-binding domain of the binding agent. In some embodiments, a mask domain of a binding agent encompassed by the present disclosure can be a fragment or domain of a receptor ligand that binds an APP-binding domain of the binding agent. In some embodiments, a mask domain of a binding agent encompassed by the present disclosure can be a small molecule that binds an APP-binding domain of the binding agent. In some embodiments, a mask domain of a binding agent encompassed by the present disclosure can be an aptamer that binds an APP-binding domain of the binding agent.

[0231] In some embodiments, a mask domain binds an APP-binding domain that is a receptor protein. In some embodiments, a mask domain binds an APP-binding domain that is a fragment or domain of a receptor protein. In some embodiments, a mask domain binds an APP- binding domain that is a receptor ligand. In some embodiments, a mask domain binds an APP- binding domain that is a fragment or domain of a receptor ligand. In some embodiments, a mask domain binds an APP-binding domain that is an antibody or antibody fragment. In some embodiments, a mask domain binds an APP-binding domain that is a small molecule. In some embodiments, a mask domain binds an APP-binding domain that is an aptamer.

[0232] In some embodiments, a mask domain binds an APP-binding domain that is a SIRPα protein. In some embodiments, a mask domain binds an APP-binding domain that is a fragment or domain of a SIRPα protein. In some embodiments, a mask domain binds an APP-binding domain that is a SIGLEC10 protein. In some embodiments, a mask domain binds an APP-binding domain that is a fragment or domain of a SIGLEC10 protein. In some embodiments, a mask domain binds an APP- binding domain that is a PD-1 protein. In some embodiments, a mask domain binds an APP-binding domain that is a fragment or domain of a PD-1 protein. In some embodiments, a mask domain binds an APP-binding domain that is an LILRB1 protein. In some embodiments, a mask domain binds an APP-binding domain that is a fragment or domain of an LILRB1 protein.

[0233] In some embodiments, a mask domain binds an APP-binding domain that can include at least one immunoglobulin heavy chain and / or at least one immunoglobulin light chain. In someembodiments, a mask domain binds an APP-binding domain that can include at least one immunoglobulin heavy chain variable domain and / or at least one immunoglobulin light chain variable domain. In some embodiments, a mask domain binds an APP-binding domain that can include CDR1, CDR2, and CDR3 of at least one immunoglobulin heavy chain variable domain and / or CDR1, CDR2, and CDR3 of at least one immunoglobulin light chain variable domain. In some embodiments, mask domains that bind an APP-binding domain can be or include an extracellular domain (ECD) of a cell surface-expressed protein, a mutated version (variant) of the ECD of the cell surface-expressed protein that is engineered to improve binding to target, intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies® minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies,, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, KALBITOR®s, CARs, engineered TCRs, and antigen- binding fragments of any of the above of any of the foregoing. 1. SIRPα binding domains

[0234] Provided herein are SIRPα-binding domains. In some embodiments, a SIRPα binding domain can be used as a mask domain in provided constructs. In embodiments herein, the SIRPα- binding domains are VHH-containing molecules comprising at least one VHH domain that specifically binds to SIRPα. In some embodiments, the VHH domain binds human SIRPα. In some embodiments, the VHH domain binds one or both alleles of the wild-type human SIRPα. In some embodiments, the VHH domains binds a variant SIRPα comprising one or more amino acid modifications compared to a wild-type SIRPα. In some embodiments, the VHH domains binds a variant SIRPα that has higher affinity for CD47 than the wild-type SIRPα.

[0235] Wildtype and various engineered variants of SIRPα are known and can be used as the APP-binding domain in binding agent constructs described herein, including any as described in Section II.A. In some embodiments, the VHH domain binds the variant high affinity CV1 SIRPα (described in Weiskopf K, et al. Science.2013; Ho CC, et al. JBC.2015; corresponding to mutations V6I, V27I (or A27I), I31F, E47V, K53R, E54Q, H56P, S66T (or L66T), V92I, based on amino acid numbering of SEQ ID NO:103 or SEQ ID NO:104). In some embodiments, the VHH domains binds a variant SIRPα that has higher affinity to CD47 than the wild-type SIRPα but lower affinity than the CV1 SIRPα variant. In some embodiments, the VHH domain binds a variant SIRPα with an E54Q mutation. In some of any of the provided embodiments, the VHH domain is pan-reactive and is able to bind to a wild-type SIRPα allele and one or more variants, including CV1 or a variant with anE54Q mutation. In some of any of the provided embodiments, the VHH domain binds SIRPα having the sequence set forth in any one of SEQ ID NOs: 10, 27, 99 or 109. In some of any of the provided embodiments, the VHH domain binds SIRPα having the sequence set forth in any one of SEQ ID NOs: 103, 104, 105, or 108.

[0236] In some embodiments, a VHH domain is an antibody fragment that is a single monomeric variable antibody domain that is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, VHH domains (also called single-domain antibodies) are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain).

[0237] Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, alpaca, vicuna, guanaco, shark, goat, rabbit, and / or bovine. In some embodiments, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca, vicuna and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the disclosure.

[0238] Methods for the screening of VHH domains, including VHH-binding polypeptides, that possess the desired specificity for SIRPα include, but are not limited to, enzyme linked immunosorbent assay (ELISA), enzymatic assays, flow cytometry, and other immunologically mediated techniques known within the art.

[0239] Among the provided VHH domains provided herein are SIRPα (human-derived ), such as any described below.

[0240] In some embodiments, a VHH domain that binds SIRPα may be derived from a non- human species and be humanized. Humanized antibodies (such as VHH-containing polypeptides) are useful as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies, which can result in an immune response to an antibody therapeutic, and decreased effectiveness of the therapeutic. Generally, a humanized antibody comprises one or more variable domains in which CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (for example, the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0241] Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci.13: 1619-1633, and are further 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 (describing “resurfacing”); Dall'Acqua et al., (2005) Methods 36:43-60 (describing “FR shuffling”); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing the “guided selection” approach to FR shuffling).

[0242] Human framework regions that can be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, for example, Sims et al. (1993) J. Immunol.151 :2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of heavy chain variable regions (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 (somatically mutated) framework regions or human germline framework regions (see, for example, Almagro and Fransson, (2008) Front. Biosci.13:1619-1633); and framework 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. 271 :22611-22618). Typically, the FR regions of a VHH are replaced with human FR regions to make a humanized VHH. In some embodiments, certain FR residues of the human FR are replaced in order to improve one or more properties of the humanized VHH. VHH domains with such replaced residues are still referred to herein as “humanized.”

[0243] Provided herein are VHH domains that bind SIRPα (SIRPα-binding VHH domain or SIRPα VHH domain) in which the VHH domain comprises a CDR1, CDR2 and CDR3 contained in a VHH amino acid sequence selected from any of SEQ ID NO: 13-21 and 28-36, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VHH amino acid sequence selected from any one of SEQ ID NOs: 13-21 and 28-36. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in any one of SEQ ID NOS: 37, 38, 39, 40, 41, 42, 43, 44, and 45, a CDR2 set forth in any one of SEQ ID NOS: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 and a CDR3 set forth in any one of SEQ ID NOS: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. Among the provided SIRPα VHH domains is a SIRPα VHH domain that has the amino acid sequence set forth in any of SEQ ID NOS:13-21 and 28-36 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VHH amino acid sequence selected from any one of SEQ ID NOs: 13-21 and 28-36. In some embodiments, the SIRPα VHH domain has the sequence of amino acids set forth in any one of SEQ ID NOS: 13-21 and 28-36.

[0244] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:13, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO:13. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:13 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:13.

[0245] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:14, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO:14. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:14 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:14.

[0246] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:15, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:15 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:15.

[0247] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:16, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:16 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:16.

[0248] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:17, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO:17. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:17 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:17.

[0249] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:18, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO:18. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:18 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:18.

[0250] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:19, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO:19. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:19 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:19.

[0251] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:20, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:20 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:20.

[0252] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:21, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the SIRPα VHH domain hasthe amino acid sequence set forth in SEQ ID NO:21 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 21.

[0253] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:28, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:28.

[0254] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO: 29, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:29.

[0255] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:30, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 30 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO:30.

[0256] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:31, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 31 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 31.

[0257] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:32, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 32.

[0258] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO: 33, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 33 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 33.

[0259] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO: 34, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 34 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 34.

[0260] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO: 35, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 35 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 35.

[0261] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1, CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO: 36, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the SIRPα VHHdomain has the amino acid sequence set forth in SEQ ID NO: 36 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the SIRPα VHH domain has the amino acid sequence set forth in SEQ ID NO: 36.

[0262] In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 37, 46 and 61, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 38, 46 and 61, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 39, 47 and 62, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 40, 48 and 63, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 41, 49 and 64, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 37, 50 and 61, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 42, 51 and 65, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 43, 52 and 66, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 37, 53 and 67, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 44, 54 and 68, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 43, 55 and 63, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 40, 56 and 69, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 37, 57 and 70, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 40, 55 and 63, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 41, 58 and 71, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 43, 59 and 72, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 37, 60 and 73, respectively. In some embodiments, the SIRPα VHH domain provided herein comprises a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 45, 56 and 73, respectively.

[0263] In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 37, a CDR2 set forth in SEQ ID NO: 46 and a CDR3 set forth in SEQ ID NO:61. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 38, a CDR2 set forth in SEQ ID NO: 46 and a CDR3 set forth in SEQ ID NO:61. Insome embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 39, a CDR2 set forth in SEQ ID NO: 47 and a CDR3 set forth in SEQ ID NO:62. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 40, a CDR2 set forth in SEQ ID NO: 48 and a CDR3 set forth in SEQ ID NO:63. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 41, a CDR2 set forth in SEQ ID NO: 49 and a CDR3 set forth in SEQ ID NO: 64. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 37, a CDR2 set forth in SEQ ID NO: 50 and a CDR3 set forth in SEQ ID NO:61. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 42, a CDR2 set forth in SEQ ID NO: 51 and a CDR3 set forth in SEQ ID NO: 65. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 43, a CDR2 set forth in SEQ ID NO: 52 and a CDR3 set forth in SEQ ID NO:66. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 37, a CDR2 set forth in SEQ ID NO: 53 and a CDR3 set forth in SEQ ID NO: 67. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 44, a CDR2 set forth in SEQ ID NO: 54 and a CDR3 set forth in SEQ ID NO:68. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 43, a CDR2 set forth in SEQ ID NO: 55 and a CDR3 set forth in SEQ ID NO:63. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 40, a CDR2 set forth in SEQ ID NO: 56 and a CDR3 set forth in SEQ ID NO: 69. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 37, a CDR2 set forth in SEQ ID NO: 57 and a CDR3 set forth in SEQ ID NO: 70. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 40, a CDR2 set forth in SEQ ID NO: 55 and a CDR3 set forth in SEQ ID NO: 63. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 41, a CDR2 set forth in SEQ ID NO: 58 and a CDR3 set forth in SEQ ID NO: 71. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 43, a CDR2 set forth in SEQ ID NO: 59 and a CDR3 set forth in SEQ ID NO: 72. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 37, a CDR2 set forth in SEQ ID NO: 60 and a CDR3 set forth in SEQ ID NO: 73. In some embodiments, a SIRPα VHH domain provided herein comprises a CDR1 set forth in SEQ ID NO: 45, a CDR2 set forth in SEQ ID NO: 56 and a CDR3 set forth in SEQ ID NO: 73.

[0264] In some embodiments, a SIRPα VHH domain is used as a mask domain in any of the providing multispecific antigen binding constructs, such as to mask a CD47 APP binding domain.

[0265] In some embodiments, a SIRPα VHH domain is used as a component of a Bispecific Macrophage Engager (BiME). In some embodiments, provided are bispecific molecules that include a SIRPα VHH domain bind to SIRPα and can block the interaction of CD47 on one cell with SIRPα on a phagocytic cell, and a second antigen Antibodies that are bispecific for SIRPα and a secondantigen are termed Bi-specific Macrophage Enhancing (BiME) antibodies. In some embodiments, such binding molecules are able to trigger phagocytic killing and immune response against a target cell, such as a tumor cell. In some embodiments, the bispecific binding molecule is able to bridge two cells (an effector and a target), in close proximity, such that other cellular receptors and membrane components on either cell can interact and the effector myeloid cell can thereby trigger engulfment of the target cell.

[0266] In some embodiments, the bispecific antibodies are directed against SIRPα and a second antigen. As such, in some cases, a subject antibody is a bispecific or multispecific antibody that specifically binds to SIRPα and at least a second antigen. The second antigen can include any tumor-associated antigen. Exemplary tumor-associated antigens include any as described in Table 2. Other exemplary second antigens are any cancer cell marker, 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, HAVCR2 (TIM3). In some cases, an exemplary bispecific antibody includes a SIRPα VHH domain sequence (e.g., CDRs) disclosed herein that provides specific binding to SIRPα as well as sequences (e.g., CDRs) from antibodies that bind a cancer cell marker. Examples of antibodies with CDRs that provide specific binding to a cancer cell marker include any as described herein. IN some embodiments, the antibody directed against a cancer marker is CETUXIMAB (binds EGFR), PANITUMUMAB (binds EGFR), RITUXIMAB (binds CD20), TRASTUZUMAB (binds HER2), PERTUZUMAB (binds HER2), ALEMTUZUMAB (binds CD52), or BRENTUXIMAB (binds CD30). (i) Exemplary features

[0267] SIRPα VHH domains provided herein may be identified, screened for, or characterized for their physical / chemical properties and / or biological activities by various known assays.

[0268] In some embodiments, the SIRPα VHH domains have one or more specified functional features, such as binding properties, including binding to SIRPα, such as a wild-type SIRPα and / or a variant SIRPα. In some embodiments, the SIRPα VHH domains are capable of binding SIRPα with at least a certain affinity. In some embodiments, the provided SIRPα VHH domains bind with moderate to high affinity for to particular epitopes, such as epitope(s) of SIRPα. In some embodiments, binding of a SIRPα VHH domain, such as binding of a SIRPα VHH domain to SIRPα. is measured by any of a number of known methods. Binding affinity may be measured as KD, KA or EC50. In some embodiments, the affinity is represented by an equilibrium dissociation constant (KD). In some embodiments, the affinity is represented by EC50.

[0269] A variety of assays are known for assessing binding affinity, equilibrium dissociation constant (KD), equilibrium association constant (KA), EC50, on-rate (association rate constant; kon or ka; units of 1 / Ms or M-1s-1) and the off-rate (dissociation rate constant; koff or kd; units of 1 / s or s-1)and / or determining whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand (e.g., an antigen). One can determine the binding affinity of a binding molecule, such as by using any of a number of binding assays that are well known. For example, 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 fragment thereof, and an antigen), using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res.53:2560, 1993). In one aspect, the SIRPα VHH domain is tested for SIRPα binding activity, e.g., by known methods. In some embodiments, binding of the SIRPα VHH domain to an antigen, such as SIRPα, such as a wild type SIRPα or variant SIRPα, is assessed by methods known in the art, such as ELISA, Western blotting, flow cytometric assays, and / or surface plasmon resonance (SPR).

[0270] In some embodiments, the equilibrium dissociation constant (KD) of the SIRPα VHH domain to SIRPα is from at or about 0.1 nM to at or about 10 µM, from at or about 0.1 nM to at or about 50 nM, from at or about 0.1 nM to at or about 40 nM, from at or about 0.1 nM to at or about 30 nM, from at or about 0.1 nM to at or about 20 nM, from at or about 0.1 nM to at or about 10 nM, from at or about 0.1 nM to at or about 1 nM, from at or about 1 nM to at or about 500 nM, from at or about 1 nM to at or about 50 nM, from at or about 1 nM to at or about 40 nM, from at or about 1 nM to at or about 30 nM, from at or about 1 nM to at or about 20 nM, from at or about 1 nM to at or about 10 nM, from at or about 10 nM to at or about 500 nM, from at or about 10 nM to at or about 50 nM, from at or about 10 nM to at or about 40 nM, from at or about 10 nM to at or about 30 nM, or from at or about 10 nM to at or about 20 nM. In certain embodiments, the binding affinity (EC50) and / or the equilibrium dissociation constant (KD) of the VHH domain to SIRPα, is at or about or less than at or 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 foregoing.

[0271] In some embodiments, the SIRPα VHH domain binds to SIRPα with a sub-nanomolar binding affinity, for example, with a binding affinity less than at or about 1 nM, such as less than at or 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, KD, of the binding molecule, e.g., SIRPα VHH domain, to SIRPα, such as a wild type or variant SIRPα, is from at or about 0.01 nM to about 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 certain embodiments, the equilibrium dissociation constant, KD, of the binding molecule, e.g., SIRPα VHH domain, to SIRPα, is at or about or less than at or about 10 μM, 5 μMm 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less, or a range defined by any of the foregoing.

[0272] In some embodiments, the provided SIRPα VHH domains bind to SIRPα with a binding affinity that allows spontaneous release of the SIRPα VHH domains from the SIRPα after cleavage of the protease cleavable linker. In some embodiments, to facilitate anti-phagocytic activity of a provided multispecific binding construct, the value of the koff of a provided SIRPα VHH domain cannot be too low and needs to be higher than that of the wild-type SIRPα or variant SIRPα of the construct toward wild-type human CD47, such as a cell surface-expressed CD47. Furthermore, the wild-type SIRPα or variant SIRPα of the construct should bind to the wild-type human CD47, such as a cell surface-expressed CD47, faster than the cleaved and dissociated mask rebinds, such that the kon value for the SIRPα VHH domain should be lower than the wild-type SIRPα or variant SIRPα antigen. In some embodiments, the SIRPα VHH domains has a lower affinity, and hence a higher dissociation constant (KD), for binding the wild-type SIRPα or variant SIRPα of the construct, than the wild-type SIRPα or variant SIRPα of the construct has for binding to wildtype human CD47, such as cell-surface expressed CD47. In some embodiments, the SIRPα VHH domain binds to the wild- type human SIRPα or the variant thereof with a dissociation constant (KD) is at least 2 times, 5 times, 10 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 time greater than the KD of the wild-type human SIRPα or the variant thereof for wild-type human CD47, such as a cell-surface expressed CD47. In some embodiments, the KD of the SIRPα VHHdomain is not less than 1 nM. In such embodiments, after cleavage of a protease cleavable linker, in which the SIRPα VHH domains is linked to SIRPα via the protease cleavable linker, the SIRPα VHH domain is released from the SIRPα. In such examples, the SIRPα is no longer bound to the SIRPα VHH domain, and the SIRPα can interact with other binding partner(s), such as, for example, CD47. C. Proteolytically Cleavable Linkers

[0273] The present disclosure provides, among other things, binding agents that include a mask domain associated with a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker is present in a polypeptide that includes multiple domains provided herein (e.g., including at least an APP-binding domain and a mask domain). In some embodiments, a mask domain is positioned such that cleavage of the proteolytically cleavable linker separates the mask domain from at least one other domain, or all other domains, present in the polypeptide and / or binding agent. In this manner, the mask domain substantially inhibits the binding of the APP-binding domain to an APP target until after cleavage of the proteolytically cleavable peptide linker in the desired environment.

[0274] In some embodiments, the proteolytically cleavable linker includes a cleavage site, such as a protease cleavage site that is recognized and can be cleaved by a protease. The proteolytically cleavable linker includes an amino acid sequence that can serve as a substrate for a protease, such as an extracellular protease.

[0275] In some embodiments, a proteolytically cleavable linker can be cleaved by a human and / or biologically relevant protease (e.g., a protease expressed by one or more cells, and / or in one or more tissues, of the human body). In some embodiments, a proteolytically cleavable linker can be cleaved by an extracellular protease. In some embodiments, a proteolytically cleavable linker can be cleaved by a cell surface protease. In some embodiments, a proteolytically cleavable linker can be cleaved by an intracellular protease. In some embodiments, a proteolytically cleavable linker can be cleaved by an aminopeptidase. In some embodiments, a proteolytically cleavable linker can be cleaved by an aspartyl protease. In some embodiments, a proteolytically cleavable linker can be cleaved by a metalloprotease. In some embodiments, a proteolytically cleavable linker can be cleaved by a cysteine protease. In some embodiments, a proteolytically cleavable linker can be cleaved by a serine protease. In some embodiments, a proteolytically cleavable linker can be cleaved by a threonine protease.

[0276] In some embodiments, a proteolytically cleavable linker can be cleaved by, and / or include a motif cleaved by, a human protease selected from ABHD12 (abhydrolase domain containing 12), ABHD12B (abhydrolase domain containing 12B), ABHD13 (abhydrolase domain containing 13), ABHD17A (family with sequence similarity 108, member A1), ABHD17B (family with sequence similarity 108, member B1), ABHD17C (family with sequence similarity 108, member C1), ABHD4 (abhydrolase dom. containing 4), ABHD5 (CGI-58), ACE (angiotensin-converting enzyme 1), ACE2 (angiotensin-converting enzyme 2), ACE3P (angiotensin-converting enzyme 3), ACR (acrosin), ACY1 (aminoacylase), ACY3 (aspartoacylase-3), ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM1A (ADAM1a), ADAM2 (ADAM2 / Fertilin-b), ADAM20, ADAM21, ADAM22, ADAM23, ADAM25 (testase 2), ADAM28, ADAM29, ADAM30, ADAM32, ADAM33, ADAM3B (ADAM3B), ADAM4, ADAM4B (ADAM4B), ADAM5, ADAM6, ADAM7, ADAM8, ADAM9, ADAMDEC1 (DECYSIN), ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ADAMTS5 (ADAMTS5 / 11), ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADGB (calpain 7-like), 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-acetylglucosaminyltransferase subunit), AMZ1 (archaemetzincin-1), AMZ2 (archaemetzincin-2), ANPEP (aminopeptidase N), AOPEP (aminopeptidase O), APEH (acylaminoacyl-peptidase), ASAH1 (acid ceramidase), ASPA (aspartoacylase), ASPRV1 (DDI-related protease), ASRGL1 (glycosylasparaginase-2), ASTL (ovastacin), ATG4A (autophagin-2), ATG4B (autophagin-1), ATG4C (autophagin-3), ATG4D (autophagin-4), ATXN3 (ataxin-3), ATXN3L (ataxin-3 like), AZU1 (azurocidin), BACE1 (beta- secretase 1), BACE2 (beta-secretase 2), BAP1 (ubiquitin C-term. hydrolase BAP1), BLMH(bleomycin hydrolase), BMP1 (procollagen C-proteinase), BRCC3 (BRCC36 / BRCA2-containing complex, sub 3), C1R (complement component C1ra), C1RL (complement C1r-homolog), C1S (complement component C1sa), C2 (complement component 2), CAD (dihydroorotase), CAPN1 (calpain 1), CAPN10 (calpain 10), CAPN11 (calpain 11), CAPN12 (calpain 12), CAPN13 (calpain 13), CAPN14 (calpain 14), CAPN15 (calpain 15 / Solh protein), CAPN2 (calpain 2), CAPN3 (calpain 3), CAPN5 (calpain 5), CAPN6 (calpain 6), CAPN7 (calpain 7), CAPN8 (calpain 8), CAPN9 (calpain 9), CARD8 (caspase recruitment domain family, member 8), CASP1 (caspase-1), CASP10 (caspase- 10), CASP12 (caspase-12), CASP14 (caspase-14), CASP16P (caspase-14-like), CASP1P2 (homologue 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 form B), CELA3A (pancreatic endopeptidase E (A)), CELA3B (pancreatic endopeptidase E (B)), CFB (complement factor B), CFD (complement factor D), CFI (complement factor I), CFLAR (casper / FLIP), CLPP (endopeptidase Clp), CMA1 (chymase), CNDP1 (carnosine dipeptidase 1), CNDP2 (carnosine dipeptidase 2), COPS5 (CSN5 / JAB1), COPS6 (COPS6), CORIN (corin), 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 (vitellogenic carboxypeptidase- L), CPXM1 (carboxypeptidase X1), CPXM2 (carboxypeptidase X2), CPZ (carboxypeptidase Z), CRMP1 (dihydropyrimidinase-rel. prot.1), CTRB1 (chymotrypsin B), CTRC (chymotrypsin C), CTRL (chymopasin), CTSA (lysosomal carboxypeptidase A), CYLD (cylindromatosis protein), CYMP (chymosin), DDI1 (DNA-damage inducible protein), DDI2 (DNA-damage inducible protein 2), DERL1 (Der1-like domain family, member 1), DERL2 (Der1-like domain family, member 2), DERL3 (Der1-like domain family, member 3), DESI1 (desumoylating isopeptidase 1), DESI2 (desumoylating isopeptidase 2), DHH (desert hedgehog protein), DNPEP (aspartyl 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 (dihydropyrimidinase), DPYSL2 (dihydropyrimidinase-rel. prot.2), DPYSL3 (dihydropyrimidinase-rel. prot.3), DPYSL4 (dihydropyrimidinase-rel. prot.4), DPYSL5 (dihydropyrimidinase-rel. prot.5), ECE1 (endothelin-converting enzyme 1), ECE2 (endothelin- converting enzyme 2), ECEL1 (DINE peptidase), ECT2L (epithelial cell transforming sequence 2 oncogene-like), EIF3F (eukar. translation initiation F3SF), EIF3H (eukar. translation initiation F3SH), ELANE (neutrophil elastase), ENPEP (aminopeptidase A), EPHX1 (epoxide hydrolase), EPHX4 (epoxyde hydrolase related protein), ERAP1 (aminopeptidase PILS), ERAP2 (aminopeptidaseMAMS / L-RAP), ERMP1 (endoplasmic reticulum metallopeptidase 1), ESPL1 (separase), F10 (coagulation factor Xa), F11 (coagulation factor XIa), F12 (coagulation factor XIIa), F2 (thrombin), F7 (coagulation factor VIIa), F9 (coagulation factor IXa), FACE1 (FACE-1 / ZMPSTE24), FACE2 (FACE-2 / RCE1), FAM111A (family with sequence similarity 111, A), FAM111B (family with sequence similarity 111, B), FAP (seprase), FOLH1 (glutamate carboxypeptidase II), FREM1 (signalase-like 1), FURIN (furin), GFPT1 (Gln-fructose-6-P transamidase 1), GFPT2 (Gln-fructose-6- P transamidase 2), GFPT3 (Gln-fructose-6-P transamidase 3), GGH (gamma-glutamyl hydrolase), GGT1 (gamma-glutamyltransferase 1), GGT2 (gamma-glutamyltransferase 2), GGT6 (gamma- glutamyltransferase 6), GGT7 (gamma-glutamyltransferase-like 3), GGTLC1 (gamma- glutamyltransferase 5), GGTLC2 (gamma-glutamyltransferase m-3), GZMA (granzyme A), GZMB (granzyme B), GZMH (granzyme H), GZMK (granzyme K), GZMM (granzyme M), HABP2 (hyaluronan-binding ser-protease), HATL2 (HAT-like 2), HATL3 (HAT-like 3), HGF (hepatocyte growth factor), HGFAC (HGF activator), HM13 (presenilin homolog 3 / SPP), HP (haptoglobin-1), HPN (hepsin), HPR (haptoglobin-related protein), HSP90AA1 (heat shock 90kDa protein 1, alpha), HSP90AB1 (heat shock 90kDa protein 1, beta), HSP90B1 (heat shock protein 90kDa beta (Grp94), member 1 / tumor rejection antigen (gp96)), HTRA1 (osteoblast serine protease), HTRA2 (HTRA2), HTRA3 (HTRA3), HTRA4 (HTRA4), IDE (insulysin), IHH (indian hedgehog protein), IMMP1L (mitochondrial signal peptidase), IMMP2L (mitoc. inner membrane protease 2), INPP5E (mitochondrial proc. protease), JOSD1 (josephin-1), JOSD2 (josephin-2), KEL (Kell blood-group protein), KHNYN (KHNYN KH and NYN domain containing), KLK1 (kallikrein hK1), KLK10 (kallikrein hK10), KLK11 (kallikrein hK11), KLK12 (kallikrein hK12), KLK13 (kallikrein hK13), KLK14 (kallikrein hK14), KLK15 (kallikrein hK15), KLK2 (kallikrein hK2), KLK3 (kallikrein hK3), KLK4 (kallikrein hK4), KLK5 (kallikrein hK5), KLK6 (kallikrein hK6), KLK7 (kallikrein hK7), KLK8 (kallikrein hK8), KLK9 (kallikrein hK9), KLKB1 (plasma kallikrein), KLKBL3 (plasma- kallikrein-like 3), LACTB (beta lactamase), LAP3 (leucyl aminopeptidase), LGMN (legumain), LMLN (leishmanolysin-2), LNPEP (leucyl-cystinyl aminopeptidase), LOC440434 (cytosol alanyl aminopep.-like 1), LONP1 (PIM1 endopeptidase), LONP2 (PIM2 endopeptidase), LPA (apolipoprotein (a)), LTA4H (leukotriene A4 hydrolase), LTF (lactotransferrin; lactoferrin), LVRN (aminopeptidase Q), MALT1 (paracaspase), MASP1 (MASP1 / 3), MASP2 (MASP2), MASTIN (mastin), MBTPS1 (site-1 protease), MBTPS2 (S2P protease), MEP1A (meprin alpha subunit), MEP1B (meprin beta subunit), MEST (mesoderm-specific transcript), METAP1 (methionyl aminopeptidase I), METAP1D (MAP1D methione aminopeptidase 1D), METAP2 (methionyl aminopeptidase II), MIPEP (mitoch. Intermediate peptidase), MME (neprilysin), MMEL1 (neprilysin- 2), MMP1 (collagenase 1), MMP10 (stromelysin 2), MMP11 (stromelysin 3), MMP12 (macrophage elastase), MMP13 (collagenase 3), MMP14 (MT1-MMP), MMP15 (MT2-MMP), MMP16 (MT3- MMP), MMP17 (MT4-MMP), MMP19 (MMP19), MMP2 (gelatinase A), MMP20 (enamelysin), MMP21 (MMP21), MMP23A (MMP23A), MMP23B (MMP23B), MMP24 (MT5-MMP), MMP25(MT6-MMP), MMP26 (matrilysin-2), MMP27 (MMP27), MMP28 (epilysin), MMP3 (stromelysin 1), MMP7 (matrilysin), MMP8 (collagenase 2), MMP9 (gelatinase B), MPND (MPND), MST1 (macrophage-stimulating protein), MYSM1 (MYSM1), N4BP1 (NEDD4 binding protein 1), NAALAD2 (NAALADASE II), NAALADL1 (NAALADASE like1), NAALADL2 (NAALADASE like 2), NAPSA (napsin A), NAPSB (napsin B), NLN (neurolysin), NLRP1 (NLRP1 self-cleaving protein), NPEPL1 (aminopeptidase-like 1), NPEPPS (cytosol alanyl aminopeptidase), NRD1 (nardilysin), NRIP2 (nuclear recept. interacting prot.2), NRIP3 (nuclear recept. interacting prot.3), NSMF (nasal embryonic LHRH factor), NUP98 (nucleoporin 98), NYNRIN (NYN domain and retroviral integrase containing), OMA1 (OMA1), OSGEP (O-sialoglycoprotein endopep.), OSGEPL1 (O-sialoglycoprotein endopep. like 1), OTUB1 (otubain-1), OTUB2 (otubain-2), OTUD1 (OTU domain containing-1), OTUD3 (OTU domain containing-3), OTUD4 (Hin-1 / OTU domain containing 4), OTUD5 (OTU domain containing-5), OTUD6A (OTU domain containing-6A), OTUD6B (OTU domain containing 6B), OTUD7A (cezanne-2), OTUD7B (cezanne / OTU domain containing 7B), OVCH1 (ovochymase-like), OVCH2 (oviductin-like / ovochymase-2), PA2G4 (proliferation- association protein 1), PAMR1 (protein C-like), PAN2 (USP52), PAPPA (pappalysin-1), PAPPA2 (pappalysin-2), PARK7 (DJ-1), PARL (presenilins-assoc. rhomboid like), 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-Pro dipeptidase), PGA3 / 4 / 5 (pepsin A), PGC (pepsin C), PGPEP1 (pyroglutamyl-peptidase I), PGPEP1L (pyroglutamyl-peptidase II), PHEX (PHEX endopeptidase), PIDD1 (PIDD auto-processing protein unit 1), PIGK (hGPI8), PIP (GCDFP15), PITRM1 (pitrilysin metalloproteinase 1), PLAT (t-plasminogen activator), PLAU (u-plasminogen activator), PLG (plasminogen), PM20D2 (PM20D2 peptidase), PMPCB (mitoch. Proc. peptidase b- subunit), PPAT (Gln-PRPP amidotransferase), PPNX (Ppnx), PRCP (lysosomal Pro-X C-peptidase), PREP (prolyl oligopeptidase), PREPL (prolyl oligopeptidase-like), PROC (protein C), PROZ (protein Z), PRPF8 (PRPF8), PRSS1 (cationic trypsin), PRSS12 (neurotrypsin), PRSS16 (thymus-specific serine peptidase), PRSS2 (anionic trypsin (II)), PRSS21 (testisin), PRSS22 (brain serine proteinase 2), PRSS23 (umbelical vein proteinase), PRSS27 (marapsin), PRSS29P (implantation serine protease 2), PRSS3 (mesotrypsin), PRSS30P (intestinal serine protease 1), PRSS33 (tryptase homolog 2 / EOS), PRSS35 (similar to SPUVE), PRSS36 (polyserase-2), PRSS37 (trypsin X2), PRSS38 (marapsin 2), PRSS3P2 (trypsin C), PRSS41 (tryptase homolog 3), PRSS42 (testis serine protease 2), PRSS45 (testis serine protease 5), PRSS48 (epidermis-specific SP-like), PRSS50 (testis-specific protein tsp50), PRSS53 (polyserase-3), PRSS54 (plasma-kallikrein-like 4), PRSS55 (plasma-kallikrein-like 2), PRSS56 (protease, serine, 56), PRSS57 (complement factor D-like), PRSS8 (prostasin), PRTN3 (proteinase 3), PSEN1 (presenilin 1), PSEN2 (presenilin 2), PSMA1 (proteasome alpha 1 subunit), PSMA2 (proteasome alpha 2 subunit), PSMA3 (proteasome alpha 3 subunit), PSMA4 (proteasome alpha 4 subunit), PSMA5 (proteasome alpha 5 subunit), PSMA6 (proteasome alpha 6 subunit),PSMA7 (proteasome alpha 7 subunit), PSMA8 (proteasome alpha 8 subunit), PSMB1 (proteasome beta 1 subunit), PSMB10 (proteasome catalytic subunit 2i), PSMB11 (proteasome b subunit LMP7- like), PSMB2 (proteasome beta 2 subunit), PSMB3 (proteasome beta 3 subunit), PSMB4 (proteasome beta 4 subunit), PSMB5 (proteasome 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 (glutaminyl cyclase), QPCTL (glutaminyl cyclase 2), RBP3 (retinol binding protein 3), RELN (reelin), REN (renin), RHBDD1 (rhomboid domain containing 1), RHBDD2 (rhomboid domain containing 2), RHBDF1 (rhomboid 5 homolog 1), RHBDF2 (rhomboid 5 homolog 2), RHBDL1 (rhomboid-like protein 1), RHBDL2 (rhomboid, veinlet-like 2), RHBDL3 (rhomboid, veinlet-like 3), RNPEP (aminopeptidase B), RNPEPL1 (aminopeptidase B-like 1), SCPEP1 (serine carboxypeptidase 1), SCRN1 (secernin-1), SCRN2 (secernin-2), SCRN3 (secernin-3), SEC11A (signalase 18 kDa component), SEC11C (signalase 21 kDa component), SENP1 (sentrin / SUMO protease 1), SENP2 (sentrin / SUMO protease 2), SENP3 (sentrin / SUMO protease 3), SENP5 (sentrin / SUMO protease 5), SENP6 (sentrin / SUMO protease 6), SENP7 (sentrin / SUMO protease 7), SENP8 (sentrin / SUMO protease 8), SHH (sonic hedgehog protein), SPG7 (paraplegin), SPPL2A (presenilin homolog 5), SPPL2B (presenilin homolog 4 / SPPL2B), SPPL2C (presenilin homolog 2), SPPL3 (presenilin homolog 1 / SPPL3), SPRTN (SprT-like N-terminal domain), ST14 (matriptase), STAMBP (AMSH / STAMBP), STAMBPL1 (AMSH-LP / STAMBPL1), SUPT16H (suppressor of Ty 16 homolog), TAF2 (TBP-associated factor 2), TASP1 (taspase), TESP2 (TESP2), TESP3 (TESP3), TESSP3 (testis serine protease 3), TESSP4 (testis serine protease 4), TESSP6 (testis serine protease 6), TFR2 (transferrin receptor 2 protein), TFRC (transferrin receptor protein), THOP1 (thimet oligopeptidase), TINAG (tubulointerstitial nephritis antigen), TINAGL1 (TINAG related protein), TLL1 (mammalian tolloid-like 1 protein), TLL2 (mammalian tolloid-like 2 protein), TMPRSS11A (TMPRSS11A), TMPRSS11B (HAT-like 5), TMPRSS11D (airway-trypsin-like protease), TMPRSS11E (DESC1 protease), TMPRSS11F (HAT-like 4), TMPRSS12 (HAT-related protease), TMPRSS13 (membrane-type mosaic Ser-prot.), TMPRSS15 (enteropeptidase), TMPRSS2 (epitheliasin), TMPRSS3 (transmembrane Ser-protease 3), TMPRSS4 (transmembrane Ser-protease 4), TMPRSS5 (spinesin), TMPRSS6 (matriptase-2), TMPRSS7 (matriptase-3), TMPRSS9 (polyserase-I), TNFAIP3 (A20, TNFa-induced protein 3), TPP1 (tripeptidyl-peptidase I), TPP2 (tripeptidyl-peptidase II), TPSAB1 (tryptase alpha / beta 1), TPSB2 (tryptase beta 2), TPSD1 (tryptase delta 1), TPSG1 (tryptase gamma 1), TRAP1 (heat shock protein 75), TRHDE (TRH-degrading ectoenzyme), TRY10 (trypsin 10), TRY15 (trypsin 15), TTC28 (HetF-like), TTR (transthyretin), TYSND1 (similar to Arabidopsis Ser-prot.), 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), 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 1), VCPIP1 (VCP(p97) / p47-interacting protein), XPNPEP1 (aminopeptidase P1), XPNPEP2 (X-prolyl aminopeptidase 2), XPNPEP3 (aminopeptidase P homologue), XRCC6BP1 (ATP23 peptidase), YME1L1 (YME1-like 1), YOD1 (OTUD2 / YOD1), ZC3H12A (zinc finger CCCH-type containing 12A), ZC3H12B (zinc finger CCCH-type containing 12B), ZC3H12C (zinc finger CCCH-type containing 12C), ZC3H12D (zinc finger CCCH-type containing 12D), ZRANB1 (TRAF-binding protein domain), and / or ZUP1 (Zinc finger containing ubiquitin peptidase 1).

[0277] In some embodiments, a proteolytically cleavable linker can be cleaved by, and / or include a motif cleaved by, a human protease selected from renin, cathepsin D, cathepsin E, pepsin C, or napsin A, matrix metalloprotease (MMP), matriptase, urokinase-type plasminogen activator (uPA), a disintegrin and metalloprotease (ADAM), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), legumain, urokinase, or hepsin.

[0278] Numerous representative examples of proteolytically cleavable linker sequences are known in the art and assays for determining protein sequence cleavage by a protease are also well- known in the art (see, for example, U.S. Pat.10,259,845 and U.S. Pat. Publ.2020 / 0115461).

[0279] In some embodiments, the proteolytically cleavable linker includes a protease cleavage site that is a tumor-associated protease cleavage site recognized by a protease whose expression is specific or upregulated for a tumor cell or tumor cell environment thereof. The cleavable linker may be selected based on a protease that is produced by a tumor that is in proximity to cells that express the target and / or produced by a tumor that is co-localized in tissue with the desired target of the multispecific polypeptide constructs. There are reports in the literature of increased levels of proteases having known substrates in a number of cancers, e.g., solid tumors. See, e.g., La Rocca et al, (2004) British J. of Cancer 90(7): 1414-1421.

[0280] In some embodiments, the protease cleavage site is a cleavage site recognized by one or more enzyme 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, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, 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, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1.

[0281] In some embodiments, the protease cleavage site is a cleavage site recognized by one or more enzyme selected from the group consisting of: ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3.

[0282] In embodiments, the protease cleavage site is a matrix metalloprotease (MMP) cleavage site, a disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site, a prostate specific antigen (PSA) protease cleavage site, a urokinase-type plasminogen activator (uPA) protease cleavage site, a membrane type serine protease 1 (MT-SP1) protease cleavage site, a matriptase protease cleavage site (ST14) or a legumain protease cleavage site. In embodiments, the matrix metalloprotease (MMP) cleavage site is a MMP9 cleavage site, a MMP13 cleavage site or a MMP2 cleavage site. In embodiments, the disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site is an ADAM9 metalloprotease cleavage site, a ADAM10 metalloprotease cleavage site or a ADAM17 metalloprotease cleavage site.

[0283] In some embodiments, the proteolytically cleavable linker is a cleavable peptide. In some embodiments, the cleavable peptide is a 5-mer (i.e. peptide 5 amino acids in length), 6-mer (i.e. peptide 6 amino acids in length), 7-mer (i.e. peptide 7 amino acids in length), 8-mer (i.e. peptide 8 amino acids in length), 9-mer (i.e. peptide 9 amino acids in length), 10-mer (i.e. peptide 10 amino acids in length), 11-mer (i.e. peptide 11 amino acids in length), 12-mer (i.e. peptide 12 amino acids in length), 13-mer (i.e. peptide 13 amino acids in length), 14-mer (i.e. peptide 14 amino acids in length), 15-mer (i.e. peptide 15 amino acids in length), 16-mer (i.e. peptide 16 amino acids in length), 17-mer (i.e. peptide 17 amino acids in length), or 18-mer (i.e. peptide 18 amino acids in length).

[0284] In some embodiments, the cleavable linker contains a substrate recognition site or cleavage site for a particular protease, which is the sequence recognized by the active site of a protease that is cleaved by a protease. Typically, for example, for a serine protease, a cleavage sequence is made up of the P1-P4 and P1′-P4′ amino acids in a substrate, where cleavage occurs after the P1 position. Typically, a cleavage sequence for a serine protease is six residues in length to match the extended substrate specificity of many proteases, but can be longer or shorter depending upon the protease. Typically, the cleavable linker includes a P1-P1′ scissile bond sequence that is recognized by a protease. In some aspects, the cleavable linker is engineered to introduce a peptide bond able to be cleaved by a specific protease, for example by introducing a substrate recognition site sequence or cleavage sequence of the protease.

[0285] In some embodiments, the protease is a granzyme B, a matriptase or an MMP, such as MMP-2. In some embodiments, the cleavable linker includes a combination of two or more substrate sequences. In some embodiments, each substrate sequence is cleaved by the same protease. In some embodiments, at least two of the substrate sequences are cleaved by different proteases.

[0286] In some embodiments, the cleavable linker comprises an amino acid that is a substrate for granzyme B. In some embodiments, a 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, a 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.

[0287] In some embodiments, the substrate for 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 contains the amino acid sequence IEPDI (SEQ ID NO:218), LEPDG (SEQ ID NO:219; LEADT (SEQ ID NO:220), IEPDG (SEQ ID NO:221), IEPDV (SEQ ID NO:222), IEPDS (SEQ ID NO:223), IEPDT (SEQ ID NO:224), IEPDP (SEQ ID NO:225), LEPDG (SEQ ID NO:226) or LEADG (SEQ ID NO:227).

[0288] In some embodiments, the cleavable linker comprises an amino acid that is a substrate for matriptase. In some embodiments, the cleavable linker comprises the sequence P4QAR↓(A / V) (SEQ ID NO: 228), wherein P4 is any amino acid. In some embodiments, the cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO: 229). In some embodiments, the substrate for matriptase comprises the amino acid sequence RQAR (SEQ ID NO: 230). In some embodiments, the cleavable linker comprises the amino acid sequence RQARV (SEQ ID NO: 231).

[0289] In some embodiments, the cleavable linker comprises an amino acid that is a substrate for one or more matrix metalloproteases (MMPs). In some embodiments, the MMP is MMP-2. In some embodiments, the cleavable linker contains the general formula P3 P2 P1 ↓ P1’ (SEQ ID NO: 232), wherein 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), wherein 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 MMP comprises the amino acid sequence PAGL (SEQ ID NO: 233).

[0290] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for matriptase. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 215) and the amino acid sequence RQAR (SEQ ID NO: 230).

[0291] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for MMP. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 215) and the amino acid sequence PAGL (SEQ ID NO: 233).

[0292] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for matriptase and an amino acid sequence that is a substrate for MMP. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence RQAR (SEQ ID NO: 230) and the amino acid sequence PAGL (SEQ ID NO: 233).

[0293] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for granzyme B, an amino acid sequence that is a substrate for matriptase, and an amino acid sequence that is a substrate for MMP. In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for MMP. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 215), the amino acid sequence RQAR (SEQ ID NO: 230), and the amino acid sequence PAGL (SEQ ID NO: 233).

[0294] The cleavable linker can include any known linkers. Examples of cleavable linkers are described in Be’liveau et al. (2009) FEBS Journal, 276; U.S. published application Nos. US20160194399; US20150079088; US20170204139; US20160289324; US20160122425; US20150087810; US20170081397; 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 by reference in its entirety.

[0295] In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of TGLEADGSPAGLGRQARVG (SEQ ID NO: 234);

[0296] In some embodiments, the proteolytically cleavable linker is the cleavable peptide VHMPLGFLGPRQARVVN (SEQ ID NO:22). In some embodiments, the proteolytically cleavable linker is the cleavable peptide ISSGLLSGRSDNH (SEQ ID NO:12).

[0297] In some embodiments, an additional linker sequence can be present N- and / or C- terminal to the protease cleavable linker. In some embodiments, an additional linker sequence is present N-terminal to the protease cleavable linker. In some embodiments, an additional linker sequence is present C-terminal to the protease cleavable linker. In some embodiments, an additional linker sequence is present N-terminal the protease cleavable linker and an additional linker sequence is present C-terminal to the protease cleavable linker. The additional linker sequence or sequences are typically flexible linker sequences Particularly suitable linker sequences predominantly include amino acid residues selected from Glycine (Gly), Serine (Ser), Alanine (Ala), and Threonine (Thr). For example, the linker may contain at least 75% (calculated on the basis of the total number of residues present in the peptide linker), such as at least 80%, at least 85%, or at least 90% of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may also consist of Gly, Ser, Ala and / or Thr residues only. In some aspects, suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, a peptide linker comprises glycine residues only. In some embodiments, a peptide linker comprises glycine and serine residues only. In some embodiments, these linkers are composed predominately of the amino acids Glycine and Serine, denoted as GS-linkers herein. In some embodiments, the linker contains (GGS)n, wherein n is 1 to 5, for example 1 to 3. In particular embodiments, the linker contains the sequence (GGGGS)n (SEQ ID NO: 259), wherein n is 1 to 5, such as 1 to 3. The linker can include combinations of any of the above, such as repeats of 2, 3, 4, or 5 GS, GGS, and / or GGGGS, linkers may be combined. In some embodiments, such a linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids in length. In some embodiments, the linker is (in one-letter amino acid code): GGS or GGGGS (SEQ ID NO: 11), GGGGSGGGGS (SEQ ID NO: 9) and GGGGSGGGGSGGGGS (SEQ ID NO:23) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 24).

[0298] In some embodiments, the linker sequence between the first antigen binding domain (e.g., APP binding domain, such as wild-type SIRPα or a variant thereof) and the second antigenbinding domain (e.g., mask domain, such as anti-SIRPα VHH domain) includes a GS linker on the N- and C-terminal ends of the protease cleavable linker, i.e. a linker sequence with the sequence GS linker- protease cleavable linker – GS linker. In some embodiments, the linker includes the sequence in N- to C-terminal order: GGGGS (SEQ ID NO: 11), the protease cleavable linker, and GGGGS (SEQ ID NO: 11). In some embodiments, the linker sequence includes the sequence in N- to C- terminal order: GGGGSGGGGS (SEQ ID NO: 9), the protease cleavable linker, and GGGGSGGGGS (SEQ ID NO: 9). In some embodiments, the linker sequence includes the sequence in N- to C- terminal order: GGGGS (SEQ ID NO: 11), the protease cleavable linker, and GGGGSGGGGS (SEQ ID NO: 9). In some embodiments, the linker sequence includes the sequence in N- to C-terminal order: GGGGSGGGGS (SEQ ID NO: 9), the protease cleavable linker, and GGGGS (SEQ ID NO: 11).

[0299] In some embodiments, the total length of the linker sequence, such as a linker sequence with the sequence GS linker- protease cleavable linker -GS linker, between the first antigen binding domain (e.g., APP binding domain, such as wild-type SIRPα or a variant thereof) and the second antigen binding domain (e.g., mask domain, such as anti-SIRPα VHH domain) is no more than 50 amino acids in length. In some embodiments, the linker is 10 to 50 amino acids in length, 10 to 40 amin acids in length, 10 to 30 amino acids in length, 10 to 20 amino acids in length, 20 to 50 amino acids in length, 20 to 40 amino acids in length, 20 to 30 amino acids in length, 30 to 50 amino acids in length, 30 to 40 amino acids in length, or 40 to 50 amino acids in length. D. Target Cell Antigens and Target Cell-Binding Domains

[0300] Binding agents encompassed by the present disclosure can include at least one binding domain that binds a target cell antigen (e.g., a cancer antigen) and at least one binding domain that binds an APP. In some embodiments, the target cell antigen is expressed on a cell targeted for myeloid cell activity (e.g., direct killing and / or indirect killing). In some embodiments, binding agents encompassed by the present disclosure comprise one target cell-binding domain that recognizes a target cell antigen expressed on a cell targeted for modulation (e.g., direct killing and / or indirect killing). In some embodiments, binding agents encompassed by the present disclosure comprise two or more target cell-binding domains (e.g., 2, 3, 4, or more) that recognize a target cell antigen expressed on a cell targeted for modulation (e.g., direct killing and / or indirect killing). In some embodiments, binding agents encompassed by the present disclosure comprise two target cell- binding domains, wherein each target cell-binding domain recognizes a different target cell antigen expressed on a cell targeted for modulation (e.g., direct killing and / or indirect killing). In some embodiments, binding agents encompassed by the present 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 a cell targeted for modulation (e.g., direct killing and / or indirect killing). A variety of target cell antigens and target cell-binding domains are known in the art. In some embodiments, a target cell antigen is a microbial antigen. In some embodiments, a target cell antigen is a peptide-major histocompatibility complex (pMHC). In some embodiments, a target cell antigen is a tumor- associated antigen (TAA). In some embodiments, a target cell antigen is a tumor-specific antigen (TSA). In some embodiments, a solid tumor antigen, e.g., a TAA or TSA, is expressed by cancer cells from a solid tumor.

[0301] In some embodiments, a cancer antigen (e.g., TAA) is selected from the group of TAAs listed in the following table or derived from a target listed in the following table: Table 2* Included in Table 2 are RNA nucleic acid molecules (e.g., thymines replaced with uredines); nucleic acid molecules encoding orthologs of the encoded proteins; DNA or RNA nucleic acid sequences comprising a nucleic acid sequence 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 more identity across their full length with any nucleic acid sequence listed in Table 2, or a portion thereof; orthologs of any protein listed in Table 2; and amino acid sequences comprising an amino acid sequence 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 more identity to across their full length with any amino acid sequence listed in Table 2, or a portion thereof. Such nucleic acid or amino acid portions can have a function of the full-length nucleic acid or amino acid molecule, respectively, as described further herein.

[0302] Representative antigen-binding domains are well-known in the art (see, e.g., U.S. Pat. No.11,459,394)) and include, without limitation, antigen-binding domains obtained from 3F8 targeting GD2 ganglioside, cantuzumab targeting MUC1, 8H9 targeting B7-H3, ecromeximab targeting GD3 ganglisode, nimotuzumab targeting EGFR; necitumumab targeting EGFR, cetuximab targeting EGFR, P2X targeting EGFR, 11F6 targeting EGFR, and other anti-EGFR antibodies such as erlotinib, osirmertnib, neratinib, gefitinib, panitumumab, dacomitnib, lapatinib, mobocertinib, and vandetanib targeting EGFR; figitumumab targeting IGF1R, seribantumab targeting ERBB3, fanvotumab targeting TYRP1, lmab362 targeting Cldn18.2, duligotumab targeting HER3, adecatumumab targeting EPCAM, trastuzumab targeting HER2, girentiuximab targeting CA9, necitumumab targeting EGFR, tucotuzumab targeting EPCAM, and zatuximab targeting HER1, 5B1 targeting Ca19-9 (see e.g., PCT publication No. WO2015053871; Sawada et al. Clin Cancer Res. 2011 Mar 1; 17(5): 1024–1032), r7E3 targeting Ca19-9 (Sawada et al.), or 121SLE targeting Ca19.9 (Sawada et al.).

[0303] In embodiments of a multispecific antigen binding construct provided herein, a third antigen binding domain is a tumor targeted domain. In some embodiments of a multispecific antigen binding construct provided herein, a third antigen binding domain is an anti-EGFR antibody or binding fragment thereof.

[0304] In some embodiments, the multispecific binding construct comprises an antibody or antigen binding fragment thereof selected from 3F8 targeting GD2 ganglioside, cantuzumab targeting MUC1, 8H9 targeting B7-H3, ecromeximab targeting GD3 ganglisode, nimotuzumab targeting EGFR, necitumumab targeting EGFR, cetuximab targeting EGFR, P2X targeting EGFR, 11F6 targeting EGFR, figitumumab targeting IGF1R, seribantumab targeting ERBB3, fanvotumab targeting TYRP1, lmab362 targeting Cldn18.2, duligotumab targeting HER3, adecatumumab targeting EPCAM, trastuzumab targeting HER2, girentiuximab targeting CA9, necitumumab targeting EGFR, tucotuzumab targeting EPCAM, zatuximab targeting HER, and 5B1 targeting Ca19.9.

[0305] In some embodiments, the multispecific binding construct comprises an anti-EGFR antibody or binding fragment thereof. In some embodiments, the antigen binding domain (e.g., third antigen binding domain) binds EGFR. In some embodiments the antigen binding domain is derived from an antibody selected from the group consisting of Necitumumab (11F8), Cetuximab, Nimotuzumab and P2X. In some embodiments, the multispecific binding construct comprises an anti- EGFR antigen binding fragment that is a Fab. In some embodiments, the multispecific binding construct comprises an anti-EGFR antigen binding fragment that is a single chain variable fragment (scFv). In some embodiments, the multispecific binding construct comprises a 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.

[0306] In some embodiments, the Fab is a Fab of Necitumumab that comprises a heavy chain comprising an amino acid sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7 and a light chain comprising a least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the Fab comprises a heavy chain comprisin...

Claims

CLAIMS What is claimed is:

1. A multispecific antigen binding construct comprising: (i) a first antigen binding domain that binds to and inhibits an anti-phagocytic 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 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, wherein the linker comprising the proteolytically cleavable linker joins the second antigen binding domain to the first antigen binding domain or to 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 joined by the linker comprising the proteolytically cleavable linker.

3. The multispecific antigen binding construct of claim 1, wherein the immunoglobulin Fc region and the second antigen binding domain are joined by the linker comprising the proteolytically cleavable linker.

4. The multispecific antigen binding construct of any one of claims 1-3, 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 the 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 the APP.

5. The multispecific antigen binding construct of any one of claims 1-4, wherein the APP is selected from the group consisting of: cluster of differentiation 47 (CD47), cluster of differentiation 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 of any one of claims 1-5, wherein the first antigen binding domain 1) inhibits the interaction of the APP with a binding partner thereof on amyeloid cell or 2) inhibits the interaction of the APP with a binding partner thereof on a cell targeted for myeloid cell activity.

7. The multispecific antigen binding construct of claim 6, wherein the myeloid cell is a macrophage, a dendritic cell, a monocyte, a neutrophil, a tumor associated macrophage (TAM), a tumor infiltrating macrophage (TIM, or a myeloid-derived suppressor cell (MDSC).

8. The multispecific antigen binding construct of any one of claims 1-7, wherein binding to and inhibiting the APP by the first antigen binding domain inhibits an interaction selected from the group consisting of the interaction between CD47 and SIRPα, the interaction between CD24 and SIGLEC10, the interaction between PD-1 and a PD-1 ligand (PD-L1 or PD-L2), the interaction between an LILRB1 ligand (β 2M or MHC-I complex) and LILRB1, and the interaction between an LILRB2 ligand 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 an MHC-I complex (HLA-A, HLA-B, or HLA-C).

9. The multispecific antigen binding construct of 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 of CD47 and wildtype SIRPα, optionally wherein the first antigen binding domain binds wildtype cell-surface expressed CD47 and and inhibits the interaction of the wildtype cell-surface expressed CD47 and wildtype cell-surface expressed SIRPα.

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 cell surface-expressed protein; (b) a binding fragment of the ECD of the cell surface-expressed protein; or (c) a variant of the ECD or the binding fragment of the cell surface-expressed protein that is engineered to improve binding to the APP.

12. The multispecific antibody binding construct of claim 11, wherein the cell surface- expressed protein is a wild-type SIRPα, and the first antigen binding domain comprises (a) an ECD of the wild-type SIRPα, (b) a binding fragment of the wild-type SIRPα ; or (c) a variant of the ECD orthe binding fragment of the wild-type SIRPα that is 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 a immunoglobulin variable (V) region (domain 1) of the ECD of the cell surface-expressed protein, optionally the ECD of the wild-type SIRPα.

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

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

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

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

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

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

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

103.

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

103.

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

103.

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

104.

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

104.

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

104.

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

27. The multispecific antigen binding construct of claim 26, wherein the variant SIRPα binds to a wildtype human CD47 with a dissociation constant (KD) of less than 100 nanomolar (nM), less than 10 nM, less than 1 nM, less than 100 picomolar (pM), less than 10 pM or less than 1 pM, or any combination between any of the foregoing.

28. The multispecific antigen binding construct of claim 26 or claim 27, wherein the variant SIRPα binds to a wildtype human CD47 with a dissociation constant (KD) of less than 100nanomolar (nM), optionally from 1 nM to 100 nM, 1 nM to 75 nM, 1 nM to 50 nM, 1 nM to 25 nM, 1 nM to 10 nM, 10 nM to 100 nM, 10 nM to 75 nM, 10 nM to 50 nM, 10 nM to 25 nM, 25 nM to 100 nM, 25 nM to 75 nM, 25 nM to 50 nM, or 50 nM to 100 nM, 50 nM to 75 nM, or 75 nM to 100 nM.

29. The multispecific antigen binding construct of claim 26 or claim 27, wherein the variant SIRPα binds to a wildtype human CD47 with a dissociation constant (KD) of less than 1 nM, optionally from 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 a wildtype human CD47 with a dissociation constant (KD) of less than 100 picomolar (pM).

31. The multispecific antigen binding construct of claim 26, claim 27 or claim 30, wherein the variant SIRPα binds to a wildtype human CD47 with a dissociation constant (KD) of from 1 pM to 100 pM, optionally from 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.

32. The multispecific antigen binding construct of 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 amino acid numbering of SEQ ID NO: 103 or SEQ ID NO:

104.

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

104.

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

104.

35. The multispecific antigen binding construct of 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 numbering of SEQ ID NO:103 or SEQ ID NO:

104.

36. The multispecific antigen binding construct of 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 amino acid numbering of SEQ ID NO:103 or SEQ ID NO:

104.

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

105.

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

105.

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

105.

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

41. The multispecific antigen binding construct of 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 amino acid numbering of SEQ ID NO:103 or SEQ ID NO:

104.

42. The multispecific antigen binding construct of any one of claims 14, 15, 26-36, 40 and 41, wherein the first antigen binding domain comprises an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10.

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

10.

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

10.

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

27.

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

27.

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

27.

48. The multispecific antigen binding construct of 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, an Fab, Fv, Fav, F(ab’)2, Fab’, dsFv, Fde, sdFv, 50. The multispecific antigen binding construct of claim 48 or claim 49, wherein the first antigen binding domain is a single domain antibody that is a VHH.

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

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

208.

53. The multispecific antigen construct of any one of claims 1-10 and 48-52, wherein the first antigen binding comprises the sequence set forth in SEQ ID NO:

208.

54. The multispecific antigen binding construct of any one of claims 1-53, wherein the second antigen binding domain has a dissociation constant for binding to the first antigen binding domain that is greater than the dissociation constant of the first antigen binding domain 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 times, 5 times, 10 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 time greater than 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 the second antigen binding domain does not interfere or compete with the first antigen binding domain for binding to the APP when the cleavable linker is cleaved.

57. The multispecific antigen binding construct of any one of claims 1-56, wherein the second antigen binding domain has a dissociation constant for binding to the first antigen binding domain that is 1 nM or greater, optionally from 100 nM to 1 µM, 10 nM to 1 µM or 1 nM to 1 µM.

58. The multispecific antigen binding construct of any one of claims 1-57, wherein the second antigen binding domain has a dissociation constant for binding to the first antigen binding domain that is 1 nM or greater.

59. The multispecific antigen binding construct of any one of claims 1-58, wherein the second antigen binding domain has a dissociation constant for binding to the first antigen binding domain that is 10 nM or greater.

60. The multispecific antigen binding construct of any one of claims 1-58, wherein the second antigen binding domain has a dissociation constant for binding to the first antigen binding domain that is 100 nM or greater.

61. The multispecific antigen binding construct of any one of claims 1-58, wherein the second antigen binding domain has a dissociation constant for binding to the first antigen binding domain that is 1 µM or greater.

62. The multispecific antigen binding construct of 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 of any one of claims 1-14 and 16-62, wherein the second antigen binding domain is an anti-SIRPα antibody or 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, an Fab, Fv, Fav, F(ab’)2, Fab’, dsFv, Fde, sdFv, 65. The multispecific antigen binding construct of any one of claims 1-64, wherein the second antigen binding domain is a single domain antibody that is a VHH.

66. The multispecific antigen binding construct of claim 65, wherein the VHH is a camelid heavy chain antibody, a humanized VHH domain, an affinity maturated 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 among SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, and 45; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from among SEQ ID NOs: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from among SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.

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

69. The multispecific antigen binding construct of any of claims 65-68, wherein the VHH domain comprises the sequence of amino acids set forth in any one of SEQ ID NOs:13-21 and 28-36 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 13-21 and 28-36, and binds SIRPα.

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

71. The multispecific antigen binding construct of any one of claims 65-70, wherein the VHH domain binds to an IgV domain of a 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 pan-reactive and binds to a wild-type SIRPα and at least one variant SIRPα comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47.

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

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

27.

75. The multispecific antigen binding construct of any one of claims 71-74, wherein the anti-SIRPα antibody or antigen-binding fragment binds to (1) an IgV domain of a 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 (a) comprising one or more amino acid substitutions in the IgV domain of the wild-type SIRPα that improves binding to CD47.

76. The multispecific antigen binding construct of any one of claims 71-75, wherein the anti-SIRPα antibody or antigen-binding fragment binds 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 that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:103, optionally a wild-type human SIRPα comprising the sequence of amino acids set forth in SEQ ID NO:103.; or (ii) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:104, optionally a wild-type human SIRPα comprising the sequence of amino acids set forth in SEQ ID NO:

104.

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

104.

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

104.

79. The multispecific antigen binding construct of claim 77 or claim 78, 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 numbering of SEQ ID NO:103 or SEQ ID NO:

104.

80. The multispecific antigen binding construct of 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 amino acid numbering of SEQ ID NO:103 or SEQ ID NO:

104.

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

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

27.

83. The multispecific antigen binding construction of any one of claims 71-82, wherein the anti-SIRPα antibody or antigen-binding fragment bind to the wild-type human SIRPα or the variant thereof with a dissociation constant (KD) is at least 2 times, 5 times, 10 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 time greater than the KD of the wild-type human SIRPα or the variant thereof for wild-type human CD47, optionally wherein the CD47 is a cell-surface expressed CD47.

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

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

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

87. The multispecific antigen binding construct of claim 85 or claim 86, wherein the protease is selected from among matriptase, a matrix metalloprotease (MMP), granzyme B, and combinations thereof.

88. The multispecific antigen binding construct of any of claims 85-87, wherein the protease is matriptase.

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

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

91. The multispecific antigen binding construct of claim 90, wherein the GS linker sequence is the sequence (GGGGS)n, wherein 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).

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

94. The multispecific antigen binding construct of any one of claims 1-14 and 16-93, wherein the third antigen binding domain is an antibody or 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, an Fab, Fv, Fav, F(ab’)2, Fab’, dsFv, Fde, sdFv, or a single domain antibody (sdAb).

96. The multispecific antigen binding construct of 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 of any one or 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 a target listed in Table 2.

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

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

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

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

103. The multispecific antigen binding construct of 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 biparatopic.

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

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

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

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

109. The multispecific antigen binding construct of 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 of any one or 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 a target listed in Table 2.

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

112. The multispecific antigen binding construct of 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 of any one of claims 1-14 and 16-114, wherein the immunoglobulin Fc region is a wildtype human IgG1 Fc region.

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

98.

117. The multispecific antigen binding construct of any one of claims 1-14 and 16-116, wherein the immunoglobulin Fc region comprises the amino acid sequence set forth 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 that increase the antibody dependent cellular cytotoxicity (ADCC)-promoting activity and / or antibody dependent cellular phagocytosis (ADCP)-promoting activity of the multispecific antigen-binding construct.

119. The multispecific antigen binding construct of claim 118, wherein the variant Fc region comprises one or more amino acid mutations compared to a wildtype 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 of 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, 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 wherein one or more of the one or more amino acid mutations are one or more amino acid substitutions, further optionally wherein 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, 267E 267L, 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.

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 comprising at least 95% sequence identity to 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 set forth in SEQ ID NO:

97.

124. The multispecific antigen binding construct of any of claims 98 and 111-123, wherein the third antigen binding region is a Fab and the multispecific antigen binding construct comprises: a first polypeptide chains comprising a heavy chain variable region (VH) and a 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 a second polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of the Fab.

125. The multispecific antigen binding construct of any of claims 98 and 111-124, wherein the third antigen binding region is a Fab and the multispecific antigen binding construct comprises: a first polypeptide comprising, in N- to C- terminal order, a heavy chain variable region (VH) and a 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 a second polypeptide comprising a light chain variable region (VL) and a 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 a disulfide bond and wherein each of the second polypeptides are covalently linked to one of the first polypeptides by a disulfide bond.

127. The multispecific antigen binding construct of any one of claims 1-14 and 16-126, wherein the third antigen binding region is a Fab and the multispecific antigen binding construct comprises: a first polypeptide comprising, in N- to C-terminal order, a heavy chain variable region (VH) and heavy chain constant region (CH1) of the Fab, an immunoglobulin Fc region comprising the sequence of amino acids set forth in SEQ ID NO: 97, a first antigen binding domain comprising thesequence of amino acids set forth in SEQ ID NO: 10, a linker comprising the proteolytically cleavage linker set forth in SEQ ID NO: 12, and a second antigen binding domain comprising a sequence that has at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 13-21 and 28- 36; and a second polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of the Fab.

128. The multispecific antigen binding construct of any one of claims 1-14 and 16-126, wherein the third antigen binding region is a Fab and the multispecific antigen binding construct comprises: a first polypeptide comprising, in N- to C-terminal order, a heavy chain variable region (VH) and heavy chain constant region (CH1) of the Fab, an immunoglobulin Fc region comprising the sequence of amino acids set forth in SEQ ID NO: 97, a first antigen binding domain comprising the sequence of amino acids set forth in SEQ ID NO: 27, a linker comprising the proteolytically cleavage linker set forth in SEQ ID NO: 12, and a second antigen binding domain comprising a sequence that has at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 13-21 and 28- 36; and a second polypeptide comprising a light chain variable region (VL) and a 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 set forth in any one of SEQ ID NOS: 13-21 and 28-36.

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

131. The multispecific antigen binding construct 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 or claim 131, wherein the GS linker is GGGGS (SEQ ID NO:11), GGGGSGGGGS (SEQ ID NO: 9), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 24).

134. The multispecific antigen binding construct of of 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 a Fab derived from an antibody selected from the group consisting of Necitumumab (11F8), Cetuximab, Nimotuzumab and P2X.

136. The multispecific antigen binding construct of any one of claims 127-135, wherein the Fab comprises: (a) a heavy chain comprising an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 7 and a light chain comprising a least 95% sequence identity to SEQ ID NO: 2; (b) a heavy chain comprising an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 205 and a light chain comprising a least 95% sequence identity to SEQ ID NO: 2; (c) a heavy chain comprising an amino acid sequence comprising at least 95% sequence identity to amino acids 1-217 of SEQ ID NO: 93 and a light chain comprising at least 95% sequence identity to SEQ ID NO: 94; (d) a heavy chain comprising a sequence comprising at least 95% sequence identity to amino acids 1-221 of SEQ ID NO: 211 and a light chain comprising at least 95% sequence identity to SEQ ID NO:96; or (e) a heavy chain comprising a sequence comprising at least 95% sequence identity to amino acids 1-217 of SEQ ID NO:212 and a light chain comprising at least 95% sequence identity to SEQ ID NO:

213.

137. The multispecific antigen binding construct of any one of claims 127-136, wherein the Fab comprises: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 205 and a light chain comprising the amino acid sequence set forth 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 set forth 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 set forth 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 set forth in SEQ ID NO:

213.

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

2.

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

2.

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

2.

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

2.

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

2.

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

2.

144. The multispecific antigen binding construct of 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 comprising at least 95% sequence identity to thesequence set forth in any one of SEQ ID NOS: 125-137 and a second polypeptide chain comprising a sequence comprising at least 95% sequence identity to the sequence set forth in SEQ ID NO:

2.

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

2.

146. The multispecific antigen binding construct of any 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 linker comprising the proteolytically cleavable linker and the second antigen binding domain.

147. The multispecific antigen binding construct of any 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, in N-to C-terminal order, the third antigen binding region, the Fc region, the first antigen binding domain, the linker comprising the proteolytically cleavable linker and the second antigen binding domain.

148. The multispecific antigen binding construct of 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 replacement of amino acids EPKSC to EPKSS.

149. The multispecific antigen binding construct of claim 148, wherein the immunoglobulin Fc region comprises an amino acid sequence comprising at least 95% sequence identity to 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 set forth in SEQ ID NO:

102.

151. The multispecific antigen binding construct of of 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 an scFv derived from selected from the group consisting of Necitumumab (11F8), Cetuximab, Nimotuzumab and P2X.

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

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

207.

155. The multispecific antigen binding construct of claim 153 or claim 153, wherein the scFv comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:

207.

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

207.

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

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

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

160. The multispecific antigen binding construct of claims 158, wherein the first antigen binding domain is monovalent and the third antigen binding domain is bivalent.

161. The multispecific antigen binding construct of any of claims 1-122 and 157-160 comprising a first antigen binding domain that binds to an anti-phagocytic protein (APP), a second antigen binding domain that binds to the first antigen binding domain, a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, and a third antigen binding domain that is a target cell antigen binding domain that binds to a target cell antigen expressed on a cell targeted for myeloid cell activity, wherein the second antigen binding domain is joined to one of the first or second Fc polypeptides by a proteolytically cleavable linker.

162. The multispecific antigen binding construct of claim 161, comprising: (1) a first heavy chain comprising the first polypeptide chain of the heterodimeric Fc and the first antigen binding domain; and (2) a second heavy chain comprising the second polypeptide chain of the heterodimeric Fc, a linker comprising the proteolytically cleavable linker and the second antigen binding domain, wherein at least one or both of the first heavy chain and second heavy chain comprising the third antigen binding domain or a chain thereof.

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

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 of claims 157-164, wherein the third antigen binding domain is a Fab, and the multispecific antigen binding construct comprises: a first polypeptide comprising, in N- to C- terminal order, a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1) of the Fab, a first polypeptide of the heterodimeric immunoglobulin Fc region, and the first antigen binding domain; a second polypeptide comprising, in N- to C-terminal order, the VH and the CH1 of the Fab, a second polypeptide of the heterodimeric Fc region, the linker comprising the proteolytically cleavable linker and the second antigen binding domain; and a third polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of the Fab.

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

167. The multispecific antigen binding construct of claim 166, wherein the one or more amino acid substitution is a knob-into-hole modification or a charge mutation to increase electrostatic complementarity of the polypeptides.

168. The multispecific antigen binding construct of claim 166 or claim 167, wherein the first Fc polypeptide of the heterodimeric Fc region comprises a amino acid substitution selected fromamong Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof and the second Fc polypeptide of the heterodimeric Fc region comprises the amino acid substitution T366W, and optionally wherein the first and second Fc polypeptides further comprises a amino acid substitution of a non-cysteine residue to a cysteine residue, wherein the amino acid substitution of the first polypeptide is at one of the position Ser354 and Y349 and the amino acid substitution of the second Fc polypeptide is at the other of the position Ser354 and Y349.

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

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

101.

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

107.

172. The multispecific antigen binding construct of any of claims 157-171, comprising a first polypeptide comprising, in N- to C-terminal order, a heavy chain variable region (VH) and heavy chain constant region (CH1) of the Fab, a first immunoglobulin Fc region comprising the sequence of amino acids set forth in SEQ ID NO: 106, a first antigen binding domain comprising the sequence of amino acids set forth in SEQ ID NO: 27; a second polypeptide comprising n N- to C-terminal order, the VH and CH1 of the Fab, a second immunoglobulin Fc region comprising the sequence of amino acids set forth in SEQ ID NO: 107, a linker comprising the proteolytically cleavage linker set forth in SEQ ID NO: 12, and a second antigen binding domain comprising a sequence that has at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOS: 13-21 and 28-36; and a third polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL) of the Fab.

173. The multispecifc antigen binding construct of claim 172, wherein the second antigen binding domain comprises the sequence set forth in any one of SEQ ID NOS: 13-21 and 28-36.

174. The multispecific antigen binding construct of of 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 a Fab derived from an antibody selected from the group consisting of Necitumumab (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 comprising at least 95% sequence identity to SEQ ID NO: 7 and a light chain comprising a least 95% sequence identity to SEQ ID NO: 2; (b) a heavy chain comprising an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 205 and a light chain comprising a least 95% sequence identity to SEQ ID NO: 2; (c) a heavy chain comprising an amino acid sequence comprising at least 95% sequence identity to amino acids 1-217 of SEQ ID NO: 93 and a light chain comprising at least 95% sequence identity to SEQ ID NO: 94; (d) a heavy chain comprising a sequence comprising at least 95% sequence identity to amino acids 1-221 of SEQ ID NO: 211 and a light chain comprising at least 95% sequence identity to SEQ ID NO:96; or (e) a heavy chain comprising a sequence comprising at least 95% sequence identity to amino acids 1-217 of SEQ ID NO:212 and a light chain comprising at least 95% sequence identity to SEQ ID NO:

213.

177. The multispecific antigen binding construct of any one of claims 174-176, wherein the Fab comprises: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 205 and a light chain comprising the amino acid sequence set forth 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 set forth 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 set forth 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 set forth in SEQ ID NO:213.

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

2.

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

2.

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

2.

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

2.

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

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

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

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

186. A cell comprising the expression vector of claim 185.

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

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

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

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

191. The method of claim 190, wherein the myeloid cell is a macrophage, a dendritic cell, a monocyte, a neutrophil, a tumor associated macrophage (TAM), a tumor infiltrating macrophage (TIM) or a or a myeloid-derived suppressor cell (MDSC).

192. The method of claim 190 or 191, wherein the target cell is infected with a microbe 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 a target listed in Table 2.

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

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

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

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 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 of any one of claims 1-181 or the pharmaceutical composition of claim 189, thereby treating and / or delaying the progression of the autoimmune or inflammatory disease in the subject.

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 carditis, rheumatoid arthritis, allergic encephalitis, atopic skin disease, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylitis, type 1 diabetes mellitus, alopecia areata, vasculitis, and temporal arteritis, graft versus host disease (GVHD), asthma, COPD, eosinophilia, conjunctivitis, glomerular nephritis, autoimmune nephritis, a paraneoplastic autoimmune disease, cartilage inflammation, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis , juvenile psoriatic arthritis, a fibrotic disease, juvenile Scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, enteropathic arthritis, reactive arthritis, Reiter's Syndrome,dermatomyositis, psoriatic arthritis, Scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodossa, Wegener's granulomatosis, arteritis, ploymyalgia rheumatica, sarcoidosis, Sclerosis, primary biliary Sclerosis, 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, rhinosinusitis, rhinosinusitis with polyps, eosinophilic esophogitis, eosinophilic bronchitis, Guillain-Barre disease, thyroiditis (e.g., Grave’s disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplantation rejection, kidney damage, hepatitis C-induced vasculitis, a viral infection, a bacterial infection, or spontaneous loss of pregnancy.

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

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, cardiac dysrhythmias, endocarditis, myocarditis, eosinophilic myocarditis, valvular heart disease, congenital heart disease, or rheumatic heart disease.

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

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-Barre´ 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 domestic 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 optionally wherein the multispecific antigen binding construct is administered subcutaneously or intravenously.

211. A SIRPα-binding molecule, comprising at least one heavy chain only variable domain (SIRPα VHH domain) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from among SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, and 45; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from among SEQ ID NOs: 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from among SEQ ID NOs: 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 a CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 37, 46 and 61, respectively; SEQ ID NOs: 38, 46 and 61, respectively; SEQ ID NOs: 39, 47 and 62, respectively; SEQ ID NOs: 40, 48 and 63, respectively; SEQ ID NOs: 41, 49 and 64, respectively; SEQ ID NOs: 37, 50 and 61, respectively; SEQ ID NOs: 42, 51 and 65, respectively; SEQ ID NOs: 43, 52 and 66, respectively; SEQ ID NOs: 37, 53 and 67, respectively; SEQ ID NOs: 44, 54 and 68, respectively; SEQ ID NOs: 43, 55 and 63, respectively; SEQ ID NOs: 40, 56 and 69, respectively; SEQ ID NOs: 37, 57 and 70, respectively; SEQ ID NOs: 40, 55 and 63, respectively; SEQ ID NOs: 41, 58 and 71, respectively; SEQ ID NOs: 43, 59 and 72, respectively; SEQ ID NOs: 37, 60 and 73, respectively; or SEQ ID NOs: 45, 56 and 73, respectively.

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

214. The SIRPα-binding molecule of any of claims 211-213, wherein the at least one SIRPα VHH domain comprises the sequence of amino acids set forth in any one of SEQ ID NOs:13- 21 and 28-36 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 13-21 and 28-36, and binds SIRPα.

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

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

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

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

27.

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

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

222. The SIRPα-binding molecule of claim 221, wherein the wild-type human SIRPα, optionally the IgV domain of the wild-type human SIRPα, comprises:(i) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:103.; or (ii) an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 in the wild-type SIRPα selected from the group consisting of L4F or L4I or L4V, V6F or V6I or V6L, V27F or V27I or V27L (A27F or A27I or A27L), I31T or I31F or I31S, E47V or E47Q or E47L, K53R, E54D or E54Q or E54H, H56P or H56L or H56R, S66G or S66T or S66A (or L66G or L66T or L66A), K68R, V92F or V92I or V92L, F94I or F94L or F94V, and F103I or F103L or F103V, corresponding to amino acid numbering of SEQ ID NO: 103 or SEQ ID NO:

104.

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

104.

226. The SIRPα-binding molecule of any 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 amino acid numbering of SEQ ID NO:103 or SEQ ID NO:

104.

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

228. The SIRPα-binding molecule of any of claims 222-227, wherein variant SIRPα, optionally the IgV domain of the variant SIRPα, comprises: an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:105;an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10; or an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

27.

229. A binding molecule comprising the SIRPα-binding molecule of any one of claims 211-228 and a second binding domain that binds to 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 a target 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 of claims 229-231, wherein the binding domain that binds to a second antigen is an antibody or antigen-binding fragment.

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

235. A nucleic acid encoding the SIRPα-binding molecule of any of claims 211-228.

236. A nucleic acid encoding the binding molecule of any of claims 229-234.

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

238. A cell comprising the expression vector of claim 237.

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

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

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

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

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

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.

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