CD1d antibodies and methods of use

Abstract

Disclosed herein is a method of treating an autoimmune disease, a cancer, or an atopic disease in a subject in need thereof comprising administering an anti-CD1d antibody to the subject.

Description

C I) 11) ANTIBODIES AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 508,207, filed June 14, 2023, the contents of which are incorporated herein by reference in its entireties.REFERENCE TO SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (LVAT_014_01WO_SeqList_ST26.xml; Size: 45,077 bytes; and Date of Creation: June 10, 2024) are herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present invention relates to methods of use of single domain antibodies that are capable of binding to CD Id for medical treatment and pharmaceutical compositions comprising the same.BACKGROUND

[0004] CD Id is a member of the CD1 (cluster of differentiation 1) family of glycoproteins (including CD la, CD lb, CDlc, CD Id and CDle) expressed on the surface of various human cells, including antigen presenting cells (APC). In humans CDld is encoded by CD1D, also known as R3G1.

[0005] Cells that are activated / stimulated via CDld include the Natural Killer T-cells (NKT cells). NKT cells recognize (self or foreign) lipids and glycolipids presented by CDld molecules present on APC. CDld restricted T cells can be divided in two groups; type 1 NKT cells which express a semi-invariant TCR and strongly react to the lipid antigen a-galactosylceramide (a-GalCer), and diverse type NKT cells which express a broad TCR repertoire and respond to various (partly unknown) lipid antigens.

[0006] The interaction between (lipid-presenting) CDld and TCR triggers the release of cytokines including Thl- or Th2-like cytokines, such as interferon- gamma, tumor necrosis factor-alpha, and interleukins like IL-4, IL-5, and IL-13.

[0007] Different lipids have been shown to bind CDld molecules, including mycolic acids, diacylglycerols, and sphingolipids. An alpha-galactosylceramide, KRN7000, is the best studied ligand of the lipid-binding CDld in NKT cell activation in vitro and in vivo. Other ligands that bind CDld comprise isoglobotrihexosylceramide, (microbial-derived) glycuronosylceramides,alpha-C-galactosylceramides, threitol ceramide, and a variety of (human and non-human) glycolipids such as lysophophatidylcholine and lysosphingomyelin.

[0008] In particular there is need for such compounds that bind and / or modulate (activate or inhibit) certain biological functions that involve CDld such as, but not limited to, NKT-cell activation, cell death or annexin V binding, but not others. Such compounds may, for example, show benefit in the various diseases in which CD Id-mediated functions play a role.SUMMARY

[0009] In some aspects described herein is a method of treating an autoimmune disease or an atopic disease in a subject in need thereof comprising administering an anti-CD Id antibody to the subject, wherein the anti-CD Id antibody binds over the A’ pocket of CDld expressed on a target cell, and wherein the anti-CD Id antibody does not induce cell death of the target cell. In some embodiments of the method, binding of the antibody increases CDld presentation of phosphatidylserine on the target cell. In some embodiments of the method the anti-CD Id antibody increases T-cell immunoglobulin and mucin domain (TIM)-1, TIM-3, and / or UM-4 binding to the target cell. In some embodiments of the method the anti-CDld antibody modulates TIM-1, TIM- 3, and / or UM-4 signaling. In some embodiments of the method the anti-CDld antibody is a single domain antibody.

[0010] In some embodiments of the method the single domain antibody comprises: (a) a CDR1 sequence selected from SEQ ID NO:2, 5, and 8, a CDR2 sequence selected from SEQ ID NO: 3, 6, and 9, and a CDR3 sequence selected from SEQ ID NO: 4, 7 and 10; (b) a CDR1 sequence selected from SEQ ID NO:12, 15, and 18, a CDR2 sequence selected from SEQ ID NO: 13, 16, and 19, and a CDR3 sequence selected from SEQ ID NO: 14, 17, and 20; (c) a CDR1 sequence selected from SEQ ID NO:22, 25, and 28, a CDR2 sequence selected from SEQ ID NO: 23, 26, and 29, and a CDR3 sequence selected from SEQ ID NO: 24, 27, and 30; (d) a CDR1 sequence selected from SEQ ID NO:32, 35, and 38, a CDR2 sequence selected from SEQ ID NO: 33, 36, and 39, and a CDR3 sequence selected from SEQ ID NO: 34, 37, and 40; or (e) a CDR1 sequence selected from SEQ ID NO:42, 45 and 48, a CDR2 sequence selected from SEQ ID NO: 43, 46, and 49, and a CDR3 sequence selected from SEQ ID NO: 44, 47, and 50. In some embodiments of the method the single domain antibody comprises an amino acid sequence that it at least 90%, at least 95%, or at least 99% identical to one of SEQ ID NOs: l, 11, 21, 31, and 41. In some embodiments of the method the single domain antibody comprises or consists of the amino acidsequence of one of SEQ ID NOs: 1, 11, 21, 31, or 41. In some embodiments of the method the autoimmune disease is selected from the group consisting of inflammatory bowel disease (IBD), multiple sclerosis, rheumatoid arthritis, psoriasis, diabetes, systemic lupus erythematosus (SLE), and ankylosing spondylitis (AS). In some embodiments of the method the atopic disease is selected from allergic asthma, atopic dermatitis, allergic rhinitis, food allergy, and allergic conjunctivitis.

[0011] In some aspects disclosed herein is a method of treating cancer in a subject in need thereof, comprising administering an anti-CDld antibody to the subject, wherein the anti-CDld antibody binds over the A’ pocket of CD Id expressed on a target cell, and wherein the anti-CDld antibody does not induce cell death of the target cell and administering a second antibody that binds to phosphatidylserine in complex with beta2 glycoprotein 1 (B2GP1) to the subject. In some embodiments of the method the antibody increases CD Id presentation of phosphatidylserine on the target cell. In some embodiments of the method the anti-CDld antibody increases T-cell immunoglobulin and mucin domain (TIM)-1, TIM-3, and / or TIM-4 binding to the target cell. In some embodiments of the method the anti-CDld antibody modulates TIM-1, TIM-3, and / or TIM- 4 signaling. In some embodiments of the method the anti-CDld antibody is a single domain antibody. In some embodiments of the method the single domain antibody comprises: (a) a CDR1 sequence selected from SEQ ID NO:2, 5, and 8, a CDR2 sequence selected from SEQ ID NO: 3, 6, and 9, and a CDR3 sequence selected from SEQ ID NO: 4, 7 and 10; (b) a CDR1 sequence selected from SEQ ID NO:12, 15, and 18, a CDR2 sequence selected from SEQ ID NO: 13, 16, and 19, and a CDR3 sequence selected from SEQ ID NO: 14, 17, and 20; (c) a CDR1 sequence selected from SEQ ID NO:22, 25, and 28, a CDR2 sequence selected from SEQ ID NO: 23, 26, and 29, and a CDR3 sequence selected from SEQ ID NO: 24, 27, and 30; (d) a CDR1 sequence selected from SEQ ID NO:32, 35, and 38, a CDR2 sequence selected from SEQ ID NO: 33, 36, and 39, and a CDR3 sequence selected from SEQ ID NO: 34, 37, and 40; or (e) a CDR1 sequence selected from SEQ ID NO:42, 45 and 48, a CDR2 sequence selected from SEQ ID NO: 43, 46, and 49, and a CDR3 sequence selected from SEQ ID NO: 44, 47, and 50. In some embodiments of the method the single domain antibody comprises an amino acid sequence that it at least 90%, at least 95%, or at least 99% identical to one of SEQ ID NOs: 1, 11, 21, 31, or 41. In some embodiments of the method the single domain antibody comprises or consists of the amino acid sequence of one of SEQ ID NOs: 1, 11, 21, 31, or 41. In some embodiments of the method the second antibody is bavituximab. In some embodiments the anti-CDld antibody is administeredprior to the second antibody. In some embodiments the anti-CDld antibody and the second antibody are administered simultaneously. In some embodiments the cancer expresses CD Id. In some embodiments the cancer is selected from a myeloid malignancy, a B-cell malignancy, renal cell carcinoma, medulloblastoma, and glioma.BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG. 1 shows expression of CDld in stably transfected in MM.ls, C1R, and HeLa cells.

[0013] FIG. 2A shows annexin V binding in the presence of CDld mAb 51.1, VHH1D17, or negative control in HeLa cells, C1R cells, and MM. Is cells induced to stably express CDld. FIG. 2B shows the fold change in annexin V binding when the cells were preincubated with VHH1D17 in both C1R and MM. Is. cells induced to stably express CDld. FIG. 2C shows the increase in fold change of annexin V binding in cells that were preincubated with increasing concentrations of antiCD Id antibodies. FIG. 2D-FIG. 2E show the fold increase over time with annexin V binding in both C1R (FIG. 2D) and MM. Is cells (FIG. 2E) that were induced to stably express CDld with, and without, prior incubation with VHH1D17.

[0014] FIG. 3A-FIG. 3B show that cell viability was not affected by incubation with VHH1D17 in either C1R (FIG. 3A) and MM. Is cells (FIG. 3B) that were induced to stably express CDld. FIG. 3C shows, similarly, that cell viability was not affected across cell lines. FIG. 3D-FIG. 3E show that cell growth was not affected in either C1R (FIG. 3D) and MM. Is cells (FIG. 3E) that were induced to stably express CDld by incubating with VHH1D17.

[0015] FIG. 4A shows the binding of annexin V when the cells are incubated with nothing, VHH1D22 (blocking antibody), VHH1D17, VHH1D17 before VHH1D22 (VHH1D17 > VHH1D22), or VHH1D22 and VHH1D17 concurrently. FIG. 4B shows the fold change quantification of FIG. 4A.

[0016] FIG. 5 shows that incubating the cells with lomitapide does not interfere with the ability VHH1D17 to induce annexin V binding.

[0017] FIG. 6A shows the shRNA-mediated knockdown of the PSAP gene. FIG. 6B shows that the shRNA knockdown of PSAP interferes with the VHH1D17 inducement of annexin V binding.

[0018] FIG. 7A shows the CRISPR-Cas9-mediated knockout of the PSAP gene. FIG. 7B shows that the KO / PSAP cells were completely deficient of PSAP protein while CDld expression wasnot affected. FIG. 7C shows that in the knockout PSAP cells VHH1D17 cannot induce annexin V binding.

[0019] FIG. 8 shows that VHH1D5 does not enhance annexin V binding.

[0020] FIG. 9A shows a part of a crystal structure depicting the similarities in the binding of the VHH1D5 and VHH1D17 proteins to CDld including the binding over the A’-pocket of CDld. FIG. 9B shows how VHH1D17 protein binds CDld in the presence of sphingomyelin. FIG. 9C shows an alanine scan of the residues around the binding site and how the mutations affect binding of VHH1D17 to CDld. FIG. 9D-FIG. 9E shows that VHH1D17 does not interfere with NKT cell activation when loaded with a-galactosylceramide (a-GalCer). FIG. 9D is in NKT12 TCRs and FIG. 9E is in NKT15 TCRs.

[0021] FIG. 10A shows a crystal structure depicting the tight overlay of VHH1D5 and VHH1D17 binding to CDld. FIG. 10B-FIG.10C show a crystal structure depicting the differences between the binding of VHH1D5 and VHH1D17 to CDld. FIG. 10B shows possible steric hinderances (specifically R109) that are caused by the Y59 position in VHH1D 17 as opposed to the 159 position in VHH1D5. FIG. 10C shows the electrostatic differences between the two proteins. VHH1D5 has an overall positive surface charge depicted in blue. VHH1D17 has an overall negative surface charge depicted in red. FIG. 10D shows the change in the surface electrostatic charge as the buffer pH goes from 4.0 to 8.0. The top is VHH1D5 bound to CDld, while the bottom is VHH1D17 bound to CDld. FIG. 10E is the quantification of the binding of VHH1D17 to CDld based on the pH of the buffer.

[0022] FIG. 11 A-FIG. 1 IB show the effect of mutating the residues around the VHH1D17 binding site to CDld on annexin V binding in both C1R (FIG. 11 A) and MM. Is (FIG. 11B) cells stably induced to express CDld.

[0023] FIG. 12 A-FIG. 12B show that VHH1D17 binding does not increase cell death (as seen in FIG. 12A) or penetration (as seen as a percentage of efferocytosis in FIG. 12B). FIG. 12C shows that VHH1D17 binding does not increase penetration. This was done at 4°C to eliminate efferocytosis.

[0024] FIG. 13 A-FIG. 13B show that in the presence of VHH1D17 there is an increase in bavituximab binding to CDld. FIG. 13A shows the flow cytometry results. FIG. 13B shows the quantification of FIG. 13A. FIG. 13C shows that VHH1D17 does not increases the degranulation of NK cells as seen by the CD 107a expression. FIG. 13D shows that there is an increase in bindingof TIM-1,3, and 4 to CDld in the presence of VHH1D17 which is lost in the PSAP knockout line. FIG. 13E shows that VHH1D17 binding increases TIM-3 binding, which is lost in the presence of an anti-TIM-3 antibody or in the PSAP knock out cells. FIG. 13F is the quantification of FIG. 13D and shows the increase in TIM-1,3, and 4 binding in the presence of VHHlD17-bound CDld, which is lost in PSAP knockout cells.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0025] The term “antibody” is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen. The antigen-binding region (or antigen-binding domain) which interacts with an antigen may comprise variable regions of both the heavy and light chains of the immunoglobulin molecule or may be a single-domain antigen-binding region, e.g. a heavy chain variable region only. The constant regions of an antibody, if present, may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells and T cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation. This also includes fragments of an antibody that retain the ability to specifically bind to the antigen. Examples of binding fragments encompassed within the term "antibody" include (i) a Fab’ or Fab fragment, i.e. a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody; (ii) F(ab')2 fragments, i.e. bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; and (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv)). Such single chain antibodies are encompassed within the term antibody unless otherwise indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility. The term antibody, unless specified otherwise, also includes polyclonalantibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, and antibody fragments provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0026] Single domain antibodies comprise a single CDR1, a single CDR2 and a single CDR3. Examples of single domain antibodies are variable fragments of heavy-chain-only antibodies, antibodies that naturally do not comprise light chains, single domain antibodies derived from conventional antibodies, and engineered antibodies. Single domain antibodies may be derived from any species including mouse, human, camel, llama, shark, goat, rabbit, and cow. For example, single domain antibodies can be derived from antibodies raised in Camelidae species, for example in camel, dromedary, llama, alpaca and guanaco. Like a whole antibody, a single domain antibody is able to bind selectively to a specific antigen. Single domain antibodies may contain only the variable domain of an immunoglobulin chain, i.e. CDR1, CDR2 and CDR3 and framework regions. Such antibodies are also called Nanobody® or VHH.

[0027] The term “immunoglobulin” as used herein is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds. The term “immunoglobulin heavy chain”, “heavy chain of an immunoglobulin” or “heavy chain” as used herein is intended to refer to one of the chains of an immunoglobulin. A heavy chain is typically comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) which defines the isotype of the immunoglobulin. The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The heavy chain constant region further comprises a hinge region. Within the structure of the immunoglobulin (e.g. IgG), the two heavy chains are inter-connected via disulfide bonds in the hinge region. Equally to the heavy chains, each light chain is typically comprised of several regions; a light chain variable region (VL) and a light chain constant region (CL). Furthermore, the VH and VL regions may be subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0028] The terms “complementarity determining region” or “CDR,” as used herein, refer to the amino acid sequences within antibody variable regions which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), or a combination thereof. In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to the amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.

[0029] The term “isotype” as used herein, refers to the immunoglobulin (sub)class (for instance IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgGlm(za) and IgGlm(f) that is encoded by heavy chain constant region genes. Each heavy chain isotype can be combined with either a kappa (K) or lambda (X) light chain. An antibody of the invention can possess any isotype.

[0030] The term “parent antibody”, or “parent immunoglobulin,” is to be understood as an antibody or immunoglobulin which is identical to an antibody or immunoglobulin according to the invention, but wherein the parent antibody or parent immunoglobulin does not have one or more of the specified mutations. A “variant” or “antibody variant” or a “variant of a parent antibody” or an “immunoglobulin variant” of the present invention is an antibody molecule or immunoglobulin molecule which comprises one or more mutations as compared to a “parent antibody” or “parent immunoglobulin.” Amino acid substitutions may exchange a native amino acid for another naturally-occurring amino acid, or for a non-naturally-occurring amino acid derivative. The amino acid substitution may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three tables:Amino acid residue classes for conservative substitutionsAlternative conservative amino acid residue substitution classesAlternative Physical and Functional Classifications of Amino Acid ResiduesConservative amino acid substitutions

[0031] In the context of antibody binding to an antigen, the terms “binds” or “specifically binds” refer to the binding of an antibody to a predetermined antigen or target (e.g. CD Id) to which binding typically is with an apparent affinity corresponding to a KD of about 10'6M or less, e.g. 10'7M or less, such as about 10'8M or less, such as about 10'9M or less, about IO'10M or less, or about IO'11M or even less, e.g. when determined using flow cytometry as described in the Examples herein. Alternatively, KD values can be determined using for instance surface plasmon resonance (SPR) technology in a BIAcore T200 or bio-layer interferometry (BLI) in an Octet RED96 instrument using the antigen as the ligand and the binding moiety or binding molecule as the analyte. Specific binding means that the antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000- fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The degree with which the affinity is lower is dependent on the KD of the binding moiety or binding molecule, so that when the KD of the binding moiety or binding molecule is very low (that is, the binding moiety or binding molecule is highly specific), then the degree with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold. The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular interaction between the antigen and the binding moiety or binding molecule.

[0032] The term “full-length antibody” when used herein, refers to an antibody which contains all heavy and light chain constant and variable domains corresponding to those that are normally found in a wild-type antibody of that isotype.

[0033] The term “chimeric antibody” refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by genetic engineering. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity.

[0034] The term “humanized antibody” refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and, optionally, fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back- mutations, may be introduced to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties. Humanization of non-human therapeutic antibodies is performed to minimize its immunogenicity in man while such humanized antibodies at the same time maintain the specificity and binding affinity of the antibody of non-human origin.

[0035] The term “multispecific antibody” refers to an antibody having specificities for at least two different, such as at least three, typically non-overlapping, epitopes. Such epitopes may be on the same or on different target antigens. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. In some embodiments, a multispecific antibody may comprise one or more single-domain antibodies.

[0036] ‘ ‘Treatment” or “treating” refers to the administration of an effective amount of an antibody according to the present invention with the purpose of easing, ameliorating, arresting, eradicating (curing) or preventing symptoms or disease states. An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. An effective amount of a polypeptide, such as an antibody, may vary according to factors such as the disease stage, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. Administration may be carried out by any suitable route, but will typically be parenteral, such as intravenous, intramuscular or subcutaneous.

[0037] The antibodies disclosed herein were developed as disclosed in WO 2016 / 122320, which is incorporated herein in its entirety by reference.Treating Autoimmune diseases

[0038] In some embodiments, the present disclosure provides methods of treating an autoimmune and / or atopic disease in a subject in need thereof comprising administering an anti-CDld antibody to the subject. In some embodiments, the anti-CDld antibody binds over the A’ pocket of CDld expressed on a target cell. In some embodiments, binding of the anti-CDld antibody does not induce cell death of the target cell.

[0039] In some embodiments, the anti-CDld antibody is a single domain antibody. Exemplary anti-CDld single domain antibodies for use according to the methods described herein are provided in Table A below. See also WO 2016 / 122320, which is incorporated herein in its entirety by reference.Table A: CDld antibody sequence.

[0040] In some embodiments, the antibody for use according to the methods described herein is selected from Table A. In some embodiments, the anti-CDld antibody is an ID5, ID17, ID3, ID6, or ID8 antibody.

[0041] In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO:2, a CDR2 sequence of SEQ ID NO: 3, and a CDR3 sequence of SEQ ID NO: 4. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO:5, a CDR2 sequence of SEQ ID NO: 6, and a CDR3 sequence of SEQ ID NO: 7. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 9, and a CDR3 sequence of SEQ ID NO: 10. In some embodiments, the anti-CDld antibody comprises or consists of SEQ ID NO: 1.

[0042] In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO: 12, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 14. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 16, and a CDR3 sequence of SEQ ID NO: 17. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO: 18, a CDR2 sequence of SEQ ID NO: 19, and a CDR3 sequence of SEQ ID NO: 20. In some embodiments, the anti-CDld antibody comprises or consists of SEQ ID NO: 11.

[0043] In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO:22, a CDR2 sequence of SEQ ID NO: 23, and a CDR3 sequence of SEQ ID NO: 24. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO:25, a CDR2sequence of SEQ ID NO: 26, and a CDR3 sequence of SEQ ID NO: 27. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO: 28, a CDR2 sequence of SEQ ID NO: 29, and a CDR3 sequence of SEQ ID NO: 30. In some embodiments, the anti-CDld antibody comprises or consists of SEQ ID NO: 21.

[0044] In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO: 32, a CDR2 sequence of SEQ ID NO: 33, and a CDR3 sequence of SEQ ID NO: 34. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO:35, a CDR2 sequence of SEQ ID NO: 36, and a CDR3 sequence of SEQ ID NO: 37. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO: 38, a CDR2 sequence of SEQ ID NO: 39, and a CDR3 sequence of SEQ ID NO: 40. In some embodiments, the anti-CDld antibody comprises or consists of SEQ ID NO: 31.

[0045] In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO:42, a CDR2 sequence of SEQ ID NO: 43, and a CDR3 sequence of SEQ ID NO: 44. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO:45, a CDR2 sequence of SEQ ID NO: 46, and a CDR3 sequence of SEQ ID NO: 47. In some embodiments, the anti-CDld antibody comprises a CDR1 sequence of SEQ ID NO: 48, a CDR2 sequence of SEQ ID NO: 49, and a CDR3 sequence of SEQ ID NO: 50. In some embodiments, the anti-CDld antibody comprises or consists of SEQ ID NO: 41.

[0046] In some embodiments the binding of the single-domain antibody to CDld does not cause cell death. In some embodiments the binding of the single-domain antibody to CDld does not cause apoptosis. In some embodiments the binding of the single-domain antibody to CDld increases presentation of anionic lipids on the cell surface. In some embodiments the anionic lipid is phospholipid is phosphatidylserine (PS). In some embodiments the binding of the single-domain antibody to CDld increases binding of a UM family member to the cell. In some embodiments the TIM family member is TIM-1. In some embodiments the TIM family member is TIM-3. In some embodiments the UM family member is TIM-4. In some embodiments the increased binding of the UM family member exerts an immunomodulatory function in vivo.

[0047] In some embodiments, the autoimmune disease is selected from an inflammatory bowel disease (IBD), multiple sclerosis, rheumatoid arthritis (RA), psoriasis, diabetes, lupus, Alzheimer’s disease, and ankylosing spondylitis.

[0048] In some embodiments, the autoimmune disease is an IBD. Herein, IBD refers to a disease characterized by a chronic inflammation of the gastrointestinal tract. In some embodiments, the IBD is selected from Crohn’s disease, ulcerative colitis, and microscopic colitis.

[0049] In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments the multiple sclerosis is selected from clinically isolated syndrome, relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), and a radiologically isolated syndrome (RIS).

[0050] In some embodiments, the rheumatoid arthritis is selected from rheumatoid factor positive (Seropositive) RA, rheumatoid factor negative (Seronegative) RA, and Juvenile RA.

[0051] In some embodiments, the psoriasis is plaque psoriasis, nail psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, and erythrodermic psoriasis.

[0052] In some embodiments, the diabetes is a type 1 diabetes, gestational diabetes, maturity onset diabetes of the young (MODY), neonatal diabetes, Wolfram Syndrome, Alstrbm Syndrome, Latent Autoimmune diabetes in Adults (LADA), type 3c diabetes, steroid-induced diabetes, and cystic fibrosis diabetes.

[0053] In some embodiments, the atopic disease is selected from allergic asthma, atopic dermatitis, allergic rhinitis, food allergy, allergic conjunctivitis (e.g., seasonal allergic conjunctivitis (SAC) or perennial allergic conjunctivitis (PAC), atopic kerato conjunctivitis (AKC), giant papillary conjunctivitis (GPC) or limbal and tarsal vernal keratoconjunctivitis (VKC).Treating Cancer

[0054] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof comprising administering an anti-CDld antibody described herein and an anti-phosphatidylserine antibody. In some embodiments the anti-phosphatidylserine antibody binds to phosphatidylserine in complex with beta2 glycoprotein 1 (B2GP1). In some embodiments the anti-phosphatidylserine antibody is bavituximab.

[0055] In some embodiments, the method comprises administration of an anti-CDld antibody described herein prior to or concurrently with an anti-phosphatidylserine antibody. In some embodiments, the anti-CDld antibody is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days prior to administration of the anti-phosphatidylserine antibody.

[0056] In some embodiments the cancer is a myeloid malignancy. In some embodiments the myeloid malignancy is myeloproliferative neoplasms. In some embodiments the myeloidmalignancy is myelodysplastic disorders. In some embodiments the myeloid malignancy is chronic myelomonocytic leukemia. In some embodiments the myeloid malignancy is acute myeloid leukemia (AML).

[0057] In some embodiments the cancer is a B-cell malignancy. In some embodiments the B-cell malignancy is a diffuse large B-cell lymphoma. In some embodiments the B-cell malignancy is a follicular lymphoma. In some embodiments the B-cell malignancy is a small lymphocytic lymphoma (SLL) or a chronic lymphocytic leukemia (CLL). In some embodiments the B-cell malignancy is a mantle cell lymphoma (MCL). In some embodiments the B-cell malignancy is a marginal zone lymphoma. In some embodiments the B-cell malignancy is a Burkitt lymphoma. In some embodiments the B-cell malignancy is a Waldenstrom macroglobulinemia. In some embodiments the B-cell malignancy is a hairy cell leukemia.

[0058] In some embodiments the cancer is a renal cell carcinoma (RCC). In some embodiments the RCC is a clear cell RCC. In some embodiments the RCC is a papillary RCC. In some embodiments the RCC is a chromophobe RCC. In some embodiments the RCC is a collecting duct RCC. In some embodiments the RCC is a multilocular cystic RCC. In some embodiments the RCC is a medullary carcinoma. In some embodiments the RCC is a renal mucinous tubular. In some embodiments the RCC is a spindle cell carcinoma. In some embodiments the RCC is an RCC associated with neuroblastoma. In some embodiments the RCC is an unclassified RCC.

[0059] In some embodiments the cancer is a medulloblastoma. In some embodiments the medulloblastoma is a classical medulloblastoma. In some embodiments the medulloblastoma is a anaplastic or large cell medulloblastoma. In some embodiments the medulloblastoma is a nodular or desmoplastic medulloblastoma. In some embodiments the medulloblastoma is a medulloblastoma with extensive nodularity (MBEN).

[0060] In some embodiments the cancer is a glioma. In some embodiments the glioma is an astrocytoma. In some embodiments the glioma is a brain stem glioma. In some embodiments the glioma is an ependymoma. In some embodiments the glioma is an oligo-astrocytoma. In some embodiments the glioma is an oligodendroglioma. In some embodiments the glioma is an optic pathway glioma.Pharmaceutical compositions, dosages, modes of administration and methods of treatment

[0061] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any of the antibodies disclosed herein and a pharmaceutically acceptable excipient.

[0062] Pharmaceutically acceptable excipients that can be used in the present compositions encompass any of the standard pharmaceutical excipients, such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents. The compositions can additionally contain solid pharmaceutical excipients such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols. For examples of excipients, stabilizers, preservatives and adjuvants, see Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990). Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

[0063] A pharmaceutical composition may also include diluents, fillers, salts, buffers, detergents, stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition. Further pharmaceutically acceptable excipients and carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption-delaying agents, and the like that are physiologically compatible with a binding molecule of the present invention.

[0064] The pharmaceutically acceptable excipient may be selected from the group consisting of fillers, e.g. sugars and / or sugar alcohols, e.g. lactose, sorbitol, mannitol, maltodextrin, etc.; surfactants, e.g. sodium lauryl sulfate, Brij 96 or Tween 80; disintegrants, e.g. sodium starch glycolate, maize starch or derivatives thereof; binder, e.g. povidone, crosspovidone, polyvinylalcohols, hydroxypropylmethylcellulose; lubricants, e.g. stearic acid or its salts; flowability enhancers, e.g. silicium dioxide; sweeteners, e.g. aspartame; and / or colorants. Pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.

[0065] The pharmaceutical composition may contain excipients for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable excipients include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen sulfite); buffers (such as borate, bicarbonate, Tris HC1, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta cyclodextrin or hydroxypropyl beta cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (in one aspect, sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990).

[0066] In some embodiments the pharmaceutical compositions are provided disclosed herein are packaged as a single unit dose. In some embodiments the pharmaceutical compositions for parenteral administration are provided as ready-to-use sterile solutions. In some embodiments, the pharmaceutical compositions are provided as sterile dry soluble products, including lyophilized powders to be reconstituted with a vehicle prior to use. In some embodiments the pharmaceutical compositions provided herein are formulated for multiple dosage administration. In some embodiments the multiple dosage parenteral formulations contain one or more antimicrobial agents at either bacteriostatic or fungistatic concentrations, or both.Methods of Administration

[0067] In some embodiments the antibodies disclosed herein are administered subcutaneously. In some embodiments the antibodies disclosed herein are administered intravenously. In someembodiments the injection is a bolus injection. In some embodiments the antibodies disclosed herein are administered intramuscularly. In some embodiments the antibodies disclosed herein are administered intraarterially.

[0068] In some embodiments the administration of the antibodies disclosed herein is preferably in a “therapeutically effective amount,” this being an amount sufficient to result in a biological or medical response of a cell, tissue, system, animal, or human that is being treated. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the disease being treated as well as the subject.

[0069] In some embodiments, any of the anti-CDld antibodies disclosed herein are administered at a dose of at least 0.01 mg / kg. In some embodiments, any of the anti-CDld antibodies disclosed herein are administered at a dose of 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, and 10 mg / kg. In some embodiments, any of the anti-CDld antibodies disclosed herein are administered at a dose of at least 1 mg. In some embodiments, any of the anti-CDld antibodies disclosed herein are administered at a dose of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, and 500 mg.

[0070] In some embodiments, any of the anti-PS disclosed herein are administered at a dose of at least 0.01 mg / kg. In some embodiments, any of the anti-PS antibodies disclosed herein are administered at a dose of 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, and 10 mg / kg. In some embodiments, any of the anti-PS antibodies disclosed herein are administered at a dose of at least 1 mg. In some embodiments, any of the anti-PS antibodies disclosed herein are administered at a dose of 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, and 500 mg. In some embodiments the dosing of anti- PS antibody is between 1 mg / kg and 3 mg / kg.Example 1: Materials and MethodsCell lines

[0071] The lymphoblastic cell line C1R (wild type (WT) or stably transduced to expressed CD Id) was grown in IMDM (Gibco, Life Technologies) supplemented with 10% (v / v) fetal calf serum (HyClone, GE Healthcare), 0.05 mM P-mercaptoethanol, 100 IU ml’1sodium penicillin, 100 pg ml’1streptomycin sulphate and 2.0 mM L-glutamine (Gibco, Life Technologies). C1R.GFP cells expressing WT CDld or mutant CDld (Q62A, H68A, V72A, R79A, E83A, K86A, M87A, V147A, Q150A, T157A, W160A) were generated as previously described (Lameris, et al., Nat. Cancer. 1 (2020) 1054-1065). The multiple myeloma cell line MM. Is (WT and stably transduced to express CDld) were generated as previously described (Song et al., Clin. Cancer Res. 14 (2008) 6955-62) and grown in RPML1640 (Gibco, Life Technologies) supplemented as above. The epithelial cell line HeLa stably transduced with CDld was previously described (Brossay, et al., J. Exp. Med. 188 (1998) 1521-8) and was grown in DMEM (Gibco, Life Technologies) supplemented as above. The aPTCR-deficient T-ALL cell lines SKW-3 cells expressing the CD Id-restricted TCRs NKT12 or NKT15 and GFP were generated as previously described (Lameris, et al., Nat. Cancer. 1 (2020) 1054-1065) and grown in RPMI-1640 supplemented as described above. The lymphoblastic natural killer 92 (NK-92) cell line stably transduced with CD 16 and GFP was previously described (Snyder et al., Front. Immunol. 9 (2018) 2873) and was grown in MEM Alpha (Gibco, Life Technologies) supplemented with 12.5% FCS (v / v), 12.5% Horse Serum (v / v) (Gibco, Life Technologies), 100 IU ml’1sodium penicillin, 100 pg ml’1streptomycin sulphate and 2.0 mM L-glutamine, and 80 U / mL interleukin 2 (IL-2, Novartis, Basel, Switzerland), as described (Snyder et al., Front. Immunol. 9 (2018) 2873). CDld expression of transfectants is shown in FIG. 1.Flow cytometry and antibodies

[0072] The following antibodies and flow cytometry reagents were used: CDld-PE (clone 51.1, Thermo Fisher cat #12-0016-42 51.1), CDld-APC (clone 51.1, Biolegend cat #350308), CD3- BV421 (clone SK7, BD Biosciences cat #563798), CD14-percpCy5.5 (clone MQP9, BD Biosciences cat #562692), CD19-APC (clone SJ25C1, BD Biosciences cat #345791), CD45- AF700 (clone HI30, Biolegend cat #304024), CD56-BV510 (clone NCAM16.2, BD biosciences cat #563041), CD69-PE (clone FN50, BD Biosciences #555531), CD107a-PE (clone H4A3,Thermo Fischer, cat #12-079-41), CD163-BV421 (clone GHI / 61, BD Biosciences cat #562643), Streptavidin-APC (eBioscience cat #17-4317-82), anti-human Fc-PE (Thermo Fisher cat #12- 4998-82), Goat-anti-mouse (GaM) Fab2 APC (Santa Cruz Biotech cat #SC-3818), rabbit antiLlama VHH (RaL)-iFluor647 (Genscript, Catalog # A02019), PSAP-unconjugated (clone 4D5F4, Thermo Fisher cat # MA5-17159), bavituximab (ADCC enhanced) (Creative Biolabs cat# TAB- 175), rituximab (Amsterdam UMC pharmacy), recombinant human(rh) TIM- 1 Fc (R&D systems cat #9319-TM), rhTIM-3 Fc (R&D systems cat #2365-TM), rhTIM-4 Fc (R&D systems cat #9300-TM), 7-amino-actinomycin D (7-AAD) (Sigma cat #A9400), annexin V-FITC (VPS diagnostics, the Netherlands, cat #A700), propidium iodide (PI) (Thermo Fisher, cat #pl6063), counting beads (Thermo Fisher cat #01-1234-42), carboxyfluorescein succinimidyl ester (CFSE) (Sigma cat#21888). All antibodies were used at empirically determined dilution factors. Staining was performed at 4 degrees Celsius in PBS supplemented with 0.1% BSA and 0.02% sodium azide or annexin V-staining buffer unless otherwise stated. Analyses were performed using a FACS LSRFortessa (BD Biosciences). Data analysis was performed using Kaluza (Beckman Coulter) and FlowJo (Tree Star Inc).Generation of CD Id-specific VHHs

[0073] Human CD Id-specific VHHs were generated and screened as previously described. See R. Lameris, et al. (Immunology. 149 (2016) 111-21). Highly purified and endotoxin free anti-CDld VHH1D7 (control anti-CDld VHH; VHH1D control), VHH1D5, VHH1D17 (wild type and single amino acid mutants) and VHH1D22 were produced by UPE, the Netherlands. For detection of binding to (WT or mutant) CDld, VHH1D17 was biotinylated using NHS-D-biotin (Sigma- Aldrich) according to the manufacturer’s protocol. IxlO5ClRGFP.CDld (WT or mutant) cells were sequentially incubated with biotinylated VHH1D17 (25 pg mF1) and Streptavidin-APC, for subsequent flow cytometry analysis. Median fluorescence was normalized for GFP expression and made relative to CDld.WT. For binding of (mutant) VHH1D17, I xlO5MM.ls.CDld cells were sequentially incubated with 200 nM of the VHHs and rabbit anti-Llama VHH (RaL)-iFluor647, for subsequent flow cytometry analysis. Relative percentage of binding was calculated by dividing median fluorescence (MF) [VHHlD17.mutant] by MF [VHH1D17.WT] multiplied by 100. For detachment of VHH1D17 at different pH, I xlO5ClRCDld cells were first incubated with 100 nM VHH1D17 (pH 7.4), washed and incubated at indicated pH (citric acid buffer) for 1 h, washed (pH 7.4) and then incubated with Streptavidin-APC, for subsequent flow cytometry analysis. 1Relative percentage of binding was calculated by dividing median fluorescence (MF) [VHH1D17 indicated pH] by MF [VHH1D17 pH 7.4] multiplied by 100.Annexin V binding and viability assessment

[0074] The effect of VHH1D17 on annexin V binding and cell viability was assessed by flow cytometry. I xlO5C1R.WT, ClRCDld cells, MMls.WT, MM. Is. CD Id cells, or Hela.CDld cells were incubated with indicated concentrations of anti-CDld VHH (VHH1D17 (WT or mutant), VHH1D5, VHH1D control) or mAb anti-CDld 51.1 (5 pg ml’1, Biolegend cat# 350304) for up to 72h. After incubation cells were washed in annexin V binding buffer and briefly incubated with annexin V-FITC and PI or 7-AAD, for subsequent flow cytometry analysis. Relative viability was calculated by dividing the percentage living (PI / 7-AAD ) [condition] by percentage living [control] cells multiplied by 100. Alternatively, living cells (7-AAD') were quantified using flow cytometric counting beads, and the relative percentage of living cells (7-AAD') was calculated by dividing the absolute cell number [condition] by absolute cell number [control] multiplied by 100. Cell expansion was determined after 96h culture by manual counting (trypan blue exclusion). Median fluorescence index (MFI) was calculated by dividing median fluorescence (MF) [condition] by MF [control].

[0075] To determine whether annexin V was bound to lipids loaded in CD Id, the type 1 NKT cell blocking anti-CDld VHH1D22 (500 nM) was added to the cell culture during or after 24h incubation with VHH1D17 as seen in R. Lameris, et al. Nat. Cancer. 1 (2020) 1054-1065 and R. Lameris, et al. Immunology. 149 (2016) 111-21. In conditions where VHH1D22 was added after incubation, it was done so at 4°C and Ih prior to analyses.Inhibition of CD Id lipid loading

[0076] Lipid loading of CD Id in the endoplasmatic reticulum was inhibited by the microsomal triglyceride transfer protein (MTP) inhibitor lomitapide (Sigma cat #SML1385). I xlO5ClRCDld cells were incubated with 1 nM lomitapide (higher concentrations induced significant cell death) or DMSO control (0.005%) ± VHH1D17 (100 nM) for 24 h. After incubation cells were analyzed as described above.

[0077] To inhibit saposin mediated lipid loading of CDld in the endosomal compartment, the prosaposin (PSAP) gene was knocked down using shRNA plasmids (sureSilencing, Qiagen cat #336312). 2xl05ClRCDld cells were transfected with PSAP shRNA-plasmid and controlshRNA-plasmid using attractene transfection reagent (Qiagen cat #301005) according to the manufacturer’s instructions, followed by positive selection with hygromycin B (200 pg ml'1). Knockdown was confirmed by intracellular staining for PSAP and subsequent flow cytometry analysis. The effect of VHH1D17 on annexin V binding was analyzed as described above.

[0078] By using CRISPR-mediated genome editing, a PSAP knockout ClR.CDld cell line (ClR.CDld. KO / PSAP) was established essentially as described in F.A. Ran, et al., Nat Protoc. 8 (2013) 2281-2308. Briefly, five PSAP-specific oligonucleotides were designed using the online E-CRISP design tool (e-crisp.org / E-CRISP / index.html), annealed, and ligated into pSpCas9(BB)- 2A-Puro (PX459) (Addgene plasmid #48139), transformed into XL-10 gold E. coli cells (Agilent technologies cat #200314), sequence validated (Baseclear BV, Leiden, The Netherlands) and electroporated into ClR.CDld cells (Neon transfection system, Thermo Fisher) according to the manufacturer’s instructions. Transfected cells were positively selected using puromycin (1 pg ml' Q. Indel mutations were detected using the SURVEYOR nuclease assay followed by clonal- density dilution to obtain clonal cell lines. ClR.CDld clones containing homogeneous indel mutations were identified by sequencing. PSAP was detected by subjecting lysates of ClR.CDld cell (clones) containing equal amounts of protein to SDS-PAGE followed by immunoblotting with anti -PSAP antibody (polyclonal, Proteintech cat #10801-1 -AP) and anti-rabbit IRDye 680RD (LI- COR cat # 926-68071). Anti-GAPDH (clone 6C5, Abeam cat #8245) and anti-mouse IRDye 800CW (LI-COR cat #926-32210) was used to confirm adequate cell lysate loading. The effect of VHH1D17 on annexin V binding was analyzed as described above.TCR transfected cell stimulation

[0079] For stimulation assays of CDld(a-GalCer)-restricted NKT12 and NKT15 TCR transfected cells, l *105TCR-expressing SKW-3 cells were co-cultured overnight, with or without l *105C1R cells (either C1R WT or ClR.CDld) and VHH1D17 (1 pM), vehicle control, a-GalCer (100 ng ml'1, Avanti Polar lipids Cat #867000P) or a combination thereof. Activation of TCR-expressing cells (GFP+CD3+CD19‘) was then analyzed by assessing CD69 expression using flow cytometry.Crystallization and structure determination

[0080] Purified P2m-CDld was generated as described in R. Lameris, et al. Nat. Cancer. 1 (2020) 1054-1065 and A.R. Aricescu, et al., Acta Crystallogr. D. Biol. Crystallogr. 62 (2006) 1243-50. Crystals of the VHHlD17-CDld(endogenous) complex were grown via the hanging drop vapordiffusion method, using a protein-reservoir drop ratio of 1: 1, at a protein concentration of 5 mg ml-1 in 12% PEG 8k, 0.05 M Zn Acetate, 0.05 M MES (pH 6.2) at 20°C. Crystals of the VHHlD17-CDld(endo) complexes were soaked in cryoprotectant solutions comprised of reservoir solution containing 10 % (v / v) ethylene glycol, before being flash frozen in liquid nitrogen. Data were collected at the Australian Synchrotron at the MX2 beamline, using the EIGER X 16M detector (Dectris Ltd) as described in D. Aragao, et al., J. Synchrotron Radiat. 25 (2018) 885-891. Data were processed using the XDS software as described in W. Kabsch, XDS., Acta Crystallogr. D. Biol. Crystallogr. 66 (2010) 125-32, and scaled using Aimless as part of the CCP4i program suite as described in M.D. Winn, et al., Overview of the CCP4 suite and current developments., Acta Crystallogr. D. Biol. Crystallogr. 67 (2011) 235-42. A single complete data sets was collected of the VHHlD17-CDld(endo) complexes. Crystal structures were solved via molecular replacement using Phaser as part of the Phenix program suite P.D. Adams, et al., PHENIX: a comprehensive Python-based system for macromolecular structure solution., Acta Crystallogr. D. Biol. Crystallogr. 66 (2010) 213-21., with the structure of the CD 1 d(aGalCer)- NKT15-TCR complex (PDB code 2PO6), as described in N.A. Borg, et al., CDld-lipid-antigen recognition by the semi-invariant NKT T-cell receptor., Nature. 448 (2007) 44-9, used as models for solving CD Id, and structure of a camelid antibody fragment (Chain B, PDB code 3P0G) used as a model for solving VHH1D17. Manual adjustments of the models were conducting using the coot graphics program as described in P. Emsley, et al., Features and development of Coot., Acta Crystallogr. D. Biol. Crystallogr. 66 (2010) 486-501, and followed by maximum-likelihood refinement using Buster 2.10 as described in G. Bricogne, et al., NBUSTER version 2.10, Cambridge, United Kingdom Glob. Phasing Ltd. (2017). All molecular model representations were generated using PyMOL. BSA values were calculated using areaimol, and contact analysis conducted using the CONTACT program, both found in the CCP4i program suite as described in M.D. Winn, et al. Definition of CDR and FR regions are based on the IMGT numbering system as described in M.-P. Lefranc, et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains., Dev. Comp. Immunol. 27 (2003) 55-77.M2-like macrophages and phagocytosis assay

[0081] Monocytes were isolated from peripheral blood mononuclear cells (PBMC) using CD 14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in complete RPMI-1640 medium in the presence of 5 ng ml’1M-CSF (R&D systems #216-MC) for 5-7 days. Cultures expressing >90% CD14 and CD163 were used for experiments. To assess phagocytosis, MM. Is. CD Id cells were stained with CFSE (5 pM) and incubated overnight in the presence or absence of anti-CDld VHH (100 nM) or bortezomib (3 nM, positive control, Axxora cat# LKT- B5871). After confirmation of induction of annexin V binding, MM. Is.CDld cells were cocultured with M2-like macrophages (1: 1 effector to target ratio) for 4h on ice or 37°C after which the percentage of CD14+CFSE+ of total CD 14+ cells was determined by flow cytometryHavituximab and TIM binding

[0082] The effect of VHH1D17 on rhTIM-1, rhTIM-3 and rhHM-4, and bavituximab binding was assessed by flow cytometry. UK)5ClR.CDld cells and ClRCDld.PSAP / KO cells were incubated ± anti-CDld VHH1D17 for 24h. rhHMl,-3,-4-Fc and bavituximab were pre-incubated with anti-Fc-PE for 20 min at room temperature, blocked with normal human serum (Merck Millipore Cat # SI) for 20 min and subsequently used to stain ClRCDld cells in the presence of in annexin V buffer, for subsequent flow cytometry analysis. MFI was calculated by dividing MF [condition] by MF [control],NK-92. CD16 stimulation assay

[0083] To evaluate induction of degranulation of NK-92. CD16 cells, 5 104ClRCDld cells, whether or not pre-incubated with VHH1D17 (24 h, 100 nM), were incubated with medium, bavituximab or rituximab (indicated concentration) for 15 min, and cocultured with 5+104NK- 92. CD 16 cells for 4h in the presence of PE-labelled anti-CD107a, after which NK-92. CD Id (GFP+CD19’CD45+CD56+)were analyzed for CD107a expression.TIM-3 reporter cell assay

[0084] The effect of VHH1D17 on TIM-3 signaling was assessed by using the TIM-3 bioassay (Thaw and Use kit, Promega Cat # JA2211). 5xl04ClRCDld cells and ClRCDld.PSAP / KO cells were incubated ± anti-CD 1 d control or anti-CD 1 d VHH1 D 17 for 24h prior to co-culture with TIM-3 effector cells (that express human TIM-3 and a T cell activation driven NanoLuc luciferase reporter), for an additional 20h followed by the addition of Bio-Glo-NL luciferase assay reagent. Bioluminescence was quantitated with a luminometer, according to the manufacturer's instructions.Statistical analysis

[0085] For in vitro experiments each n represents an independent experiment. No statistical method was used to predetermine sample size for the in vitro experiments. No data were excluded from the analyses. The experiments were not randomized and the investigators were not blinded to allocation during the experiments and outcome assessment. For data with one variable and two groups, a two-tailed paired / unpaired t-test was used to calculate the P value. For data with one variable and multiple groups, a mixed effect analysis or a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test to calculate the multiplicity-adjusted P value was used. For data with two variables, a two-way ANOVA with either Tukey’s or Sidak’s multiple comparisons test to calculate the multiplicity-adjusted P value was used. Dose-response curves, half maximal effective concentration (EC50) was calculated using nonlinear regression (agonist versus response). Statistical details can be found in the figure legend. Statistical analysis was performed using Prism v.9.1.0 (GraphPad Software).Example 2: Annexin V binding and viability assessment

[0086] The effect of VHH1D17 on annexin V binding and cell viability was assessed by flow cytometry. Cells were incubated with either mAb anti-CDld clone 51.1 or VHH1D17: (1) C1R (C1R WT); (2) C1R cells that were stably transduced to express CDld (ClRCDld); (3) MM.ls (MM. Is WT); (4) MM.ls cells that were stably transduced to express CDld cells (MM. Is. CD Id); and (5) Hela cells that were stably transduced to express CDld (Hela.CDld) (as the negative control). FIG. 1 shows expression of the CDld protein in these cell lines. After incubation cells were washed in annexin V binding buffer and briefly incubated with annexin V-FITC and PI or 7- AAD for subsequent flow cytometry analysis.

[0087] VHH1D17 consistently enhanced annexin V binding to the whole population of CDld- expressing C1R cells and MM. Is cells (FIG. 2A and FIG. 2B). In contrast, enhanced annexin V binding induced by an anti-CD 1 d monoclonal antibody (clone 51.1) was only observed for a minor population of CD Id-expressing C1R cells and was not observed in CD Id-expressing MM. Is cells (FIG. 2A and FIG. 2B) Neither VHH1D17 nor CDld mAb 51.1 were able to enhance annexin V binding to CD Id-expressing HeLa cells, which are known to differ in trafficking of CDld (FIG.2A and FIG. 2B)

[0088] Antibody targeting of CDld on multiple myeloma (MM) cells was previously reported to induce apoptosis. See E. Spanoudakis, et al., Regulation of multiple myeloma survival andprogression by CDld, Blood. 113 (2009) 2498-2507. Surprisingly, while VHH1D17 enhanced annexin V binding to CDld+ C1R and MM cells, treatment with VHH1D17 only minimally affected cell viability (FIG. 3A-FIG. 3B) and cell growth (FIG. 3C). Collectively, these data show that while VHH1D17 enhances annexin V binding to CDld+ C1R and MM cells, this does not lead to (early) cell death.Example 3: VHH1D17 enhances saposin-dependent presentation of anionic phospholipids in CDld

[0089] In healthy cells lipid transporters shuttle PS to the inner leaflet of the plasma membrane and thereby maintain a highly asymmetrical lipid distribution which is lost upon (early) apoptosis. PS externalization can, however, also occur in conditions not related to cell death, e.g. in stressed and activated immune cells. See Birge et al., Cell Death Differ. 23 (2016) 962-78. Moreover, it has also been shown that a range of (anionic) phospholipids, including PS, can be presented by CDld. See Cox, et al. PLoS One. 4 (2009) e5325. Experiments were performed to determine whether the VHHlD17-enhanced annexin V binding to CDld+tumor cells could be due to increased anionic phospholipid antigen presentation in CDld.

[0090] Since CDld restricted T cells can be divided in two groups; type 1 NKT cells which express a semi-invariant TCR and strongly react to the lipid antigen a-galactosylceramide (a-GalCer), and diverse type NKT cells which express a broad TCR repertoire and respond to various (partly unknown) lipid antigens, it was first determined if binding of annexin V could be blocked by the CDld-specific VHH1D22 that binds over the side of the F'-pocket of CDld and thereby loses the type 1 NKT-cell activation. See R. Lameris et al. VHH1D22, when added 1 hr prior to the addition of annexin V during analysis, resulted in a clear reduction in the capacity of annexin V to bind to CDld+C1R cells that had been cultured in the presence of VHH1D17. Furthermore, the increase in annexin V binding to CDld+C1R cells induced by VHH1D17 was completely lost when VHH1D22 was added at the start of the 24 h culture (FIGs. 4A-4B), indicating that the observed increase in annexin V binding results from an increased interaction with CDld-anionic phospholipid complexes.

[0091] To determine whether VHH1D17 enhances the presentation of anionic phospholipids by CD 1 d, the loading of CDld on to two different lipid transfer proteins (LTPs), known to be involved with CDld, was inhibited. Lipid loading of CDld in the endoplasmatic reticulum was inhibited by the microsomal triglyceride transfer protein (MTP) inhibitor lomitapide (Sigma cat #SML1385).The inhibition of MTP using lomitapide did not negatively impact the VHHlD17-induced increase in annexin V binding. (FIG. 5).

[0092] In contrast shRNA-mediated knockdown (KD) of prosaposin (PSAP), the precursor of saposins A-D which facilitate loading of endogenous and exogenous lipids in the endosomal / lysosomal compartment after recycling of CD Id from the cell membrane, clearly reduced and delayed the effect of VHH1D17 on annexin V binding to CDld+ C1R cells (FIG. 6B). Knockdown was confirmed by intracellular staining for PSAP and subsequent flow cytometry analysis. (FIG. 6A).

[0093] As a further confirmation of the role that PSAP plays in facilitating the increase of binding of annexin V in the presence of VHH1D17, a CRISPR-Cas9-mediated PSAP knockout (KO) CDld+ClRcell line (ClR.CDldKO / PSAP) was made. KO / PSAP cells were completely deficient of PSAP protein while CD Id expression was not affected (FIG. 7A-FIG. 7B) and resulted in a complete ablation of the capacity of VHH1D17 to increase annexin V binding (FIG. 7C).

[0094] This shows that the enhanced binding of annexin V to CDld+tumor cells exposed to VHH1D17 results from increased saposin-dependent loading of anionic phospholipid antigens of CD Id in the endosomal / lysosomal compartment.Example 4: Molecular basis of the VHH1D17-CDld interaction

[0095] To analyze the molecular basis of the interaction between VHH1D17 and CDld and how this could impact lipid loading, the crystal structure of the VHHlD17-CDld (endogenous lipid) complex at 2.25 A was determined.

[0096] The crystal structure was compared to the VHHlD5-CDld(a-GalCer) structure (Protein Data Bank (PDB) code 6V7Y) as this VHH has a high sequence homology but does not enhance annexin V binding (FIG. 8).

[0097] The electron density for the endogenous lipid matched a sphingomyelin C:20. The crystal structure of VHHlD17-CDld(sphingomyelin) showed binding of VHH1D17 to the A'-pocket of CDld by a network of van der Waals (vdw) and hydrogen bond interactions which was highly similar to binding of VHH1D5 to CDld (FIG. 9A).

[0098] Upon ligation of VHH1D17 to CDld the total buried surface area (BSA) was 1790 A2, with the CDR3 loop mediating the predominant interactions with both a-helices of CDld (71% BSA) relative to the interaction provided by the CDR1 and CDR2 loops (11% and 18% BSA, respectively), which both bound the al -helix of CDld (FIG. 9B). Also, the CDR3 loop of VHH1D17 extended toward the F'-portal and formed a hydrogen bond (R115) with the phosphatemoiety of the presented sphingomyelin, a feature shared with all phospholipids, the headgroup remained exposed to the solvent (FIG. 9B). This indicates that VHH1D17 is not expected to block the lipid headgroup of phospholipids from interacting with annexin V.

[0099] Similarly, in SKW-3 cell lines transduced with the CDld(a-GalCer)-restricted NKT12 (FIG. 9D) or NKT15 (FIG. 9E) TCRs, VHH1D17 did not interfere with type 1 NKT cell activation in response to a-GalCer loaded CD Id. This indicates that the glycosyl head group, which is important for specific recognition by the type 1 NKT TCR, remained exposed.

[0100] An alanine-substitution mutant ClR.CDld-GFP reporter cell line confirmed the prominent role of the CDR3 loop of VHH1D17 by showing that the substitution of the CD Id residue W160, which strongly interacts with the CDR3 loop, abolished binding of VHH1D17 (FIG. 9C).

[0101] One possible explanation for the difference in annexin V binding can be based on the differences in binding of VHH1D5 and VHH1D17. These two structures have a tight overlay with a core root mean square deviation (RMSD) of only 0.7 A, indicating highly similar folding (FIG. 10A). A minor difference is observed in the CDR2 loop positions, whereby the D55-G55 substitution in VHH1D17 compared to VHH1D5 decreased CDR2-CDld interactions. A likely more important difference between VHH1D5 and VHH1D17 is related to the conformational differences in the CDR3 positioning; specifically remodeling of the R109. For VHH1D17, the bulky side chain of Y59 caused a conformational change in the CDR3 region, pushing R109 towards the solvent (facing, but not contacting CDld) (FIG. 10B). Whereas for VHH1D5, the smaller 159 residue and the Pl 08, which likely prevents a-helical formation due to steric hindrance, allowed R109 to flip towards the core of the VHH and form a hydrogen bond with the backbone of Y60 allowing the CDR3 region to curl upwards (FIG. 10B). This difference altered the surface architecture and electrostatics of the side of the VHHs; where VHH1D5 has an electrostatically positive surface, VHH1D17 has a (larger) slightly electrostatically negative surface (FIG. 10C). This electrostatic charge is likely maintained (based on calculated electrostatic surface potentials (using the PDB2PQR web server) at the pH of 5 (FIG. 10D) that is commonly encountered within the endosomal / lysosomal compartment. Furthermore, binding of VHH1D17 to CDld was not affected at this pH and was only reduced at pH 4.0, possibly due to heavily compromised cell viability (FIG. 10D-FIG. 10E).

[0102] Single alanine substitution mutations of the amino-acids discordant between VHH1D5 and VHH1D17 as well as a Y59I substitution (presumed involved in positioning of the CDR3 region)in VHH1D17 did not reduce the ability of VHH1D17 to enhance annexin V binding (FIG. 11A- FIG. 11B), indicating that multiple dissimilar amino acids (likely 159 and P108) are involved.

[0103] Collectively, these data demonstrate that VHH1D17 binds to the A'-pocket of CDld and that the side of VHH1D17 has an electrostatically negative charge that is not present in VHH1D5 and could be involved in the ability of VHH1D17 to enhance anionic lipid presentation by CDld.Example 4: VHH1D17 enhanced CDld-anionic phospholipid antigen presentation does not enhance efferocytosis but enhances binding to bavituximab and facilitates CDld+tumor cells to interact with immunoregulatory TIM molecules

[0104] Exposure of the anionic phospholipid PS on the outer membrane leaflet can have a strong immune modulatory function via multiple receptors, including T-cell immunoglobulin and mucin domain containing molecules (TIM), and can serve as an eat-me signal for efferocytosis by macrophages. Also, VHHlD17-enhanced anionic phospholipid presentation by CDld does not result in increased efferocytosis in monocyte derived macrophages (moMacrophage) whereas CDld+MM cells were pretreated with bortezomib, triggering actual cell death (i.e., increase in 7- AAD+cells, FIG. 12A), were engulfed (FIG. 12B). No such effect was observed at 4 °C (FIG. 12C), indicating efferocytosis and not simple binding interaction which would also still occur at a low temperature.

[0105] Since PS exposure on tumor cells serves as an immunosuppressive signal, PS blocking antibodies may restore antitumor immunity. One such antibody is bavituximab which binds to PS in complex with 02 -glycoprotein 1 (GP1) and can trigger Fcy-receptor medicated antibodydependent cellular cytotoxicity (ADCC). Binding of bavituximab to CDld+C1R cells was indeed significantly enhanced following culture with VHH1D17 (FIG. 13A-FIG. 13B). However, VHH1D17 did not increase bavituximab-mediated degranulation on the natural killer (NK) cell line NK-92 transfected with Fey receptor III (CD16) in culture with CDld+C1R cells (FIG. 13C). Since, in the absence of VHH1D17, bavituximab induced degranulation was already high and equivalent to that triggered by the anti-CD20 therapeutic antibody rituximab, it is possible that exposed PS in the ClR.CDld NK92.CD16 cocultures was already sufficient to maximize degranulation.

[0106] Experiments were then performed to determine whether VHH1D17 could enhance TIM-1, -3, and / or -4 interactions. Analogous to enhanced annexin V binding, there was a clear enhancement in binding of recombinant human (rh)TIM-l,-3,-4 to CDld expressing C1R cells(FIG.13D-FIG. 13F) but not to the KO / PSAP CDld expressing C1R cells (FIG. 13D-FIG. 13F). In a TIM-3 bioluminescence reporter cell-based assay there was an increase in bioluminescence upon co-culture of TIM-3 reporter cells and VHH1D17 (but not control CDld VHH) exposed ClR.CDld cells which could be blocked by anti-TIM-3 mAb (FIG. 13E). Importantly, the capacity of VHH1D17 to increase TIM-3 signaling was completely abolished when using the KO / PSAP CDld expressing C1R cells (FIG. 13F).

[0107] Overall, these data demonstrate that the VHH1D17 enhanced PS presentation by CDld does not facilitate effecterocytosis of CDld+tumor cells, but does allow interaction with PS- specific therapeutic antibodies as well as immunomodulatory TIM molecules.

Claims

Claims1. A method of treating an autoimmune disease or an atopic disease in a subject in need thereof comprising administering an anti-CDld antibody to the subject, wherein the anti-CDld antibody binds over the A’ pocket of CD Id expressed on a target cell, and wherein the anti- CDld antibody does not induce cell death of the target cell.

2. The method of claim 1, wherein the binding of the antibody increases CD Id presentation of phosphatidylserine on the target cell.

3. The method of claim 1 or claim 2, wherein the anti-CDld antibody increases T-cell immunoglobulin and mucin domain (TIM)-1, TIM-3, and / or TIM-4 binding to the target cell.

4. The method of any one of claims 1-3, wherein the anti-CDld antibody modulates TIM-1, TIM-3, and / or TIM-4 signaling.

5. The method of any one of claims 1-4, wherein the anti-CDld antibody is a single domain antibody.

6. The method of claim 5, wherein the single domain antibody comprises:(a) a CDR1 sequence selected from SEQ ID NO:2, 5, and 8, a CDR2 sequence selected from SEQ ID NO: 3, 6, and 9, and a CDR3 sequence selected from SEQ ID NO: 4, 7 and 10;(b) a CDR1 sequence selected from SEQ ID NO: 12, 15, and 18, a CDR2 sequence selected from SEQ ID NO: 13, 16, and 19, and a CDR3 sequence selected from SEQ ID NO: 14, 17, and 20;(c) a CDR1 sequence selected from SEQ ID NO:22, 25, and 28, a CDR2 sequence selected from SEQ ID NO: 23, 26, and 29, and a CDR3 sequence selected from SEQ ID NO: 24, 27, and 30;(d) a CDR1 sequence selected from SEQ ID NO:32, 35, and 38, a CDR2 sequence selected from SEQ ID NO: 33, 36, and 39, and a CDR3 sequence selected from SEQ ID NO: 34, 37, and 40; or(e) a CDR1 sequence selected from SEQ ID NO:42, 45 and 48, a CDR2 sequence selected from SEQ ID NO: 43, 46, and 49, and a CDR3 sequence selected from SEQ ID NO: 44, 47, and 50.

7. The method of claim 5 or claim 6, wherein the single domain antibody comprises an amino acid sequence that it at least 90%, at least 95%, or at least 99% identical to one of SEQ ID NOs: 1, 11, 21, 31, and 41.

8. The method of claim 5 or claim 6, wherein the single domain antibody comprises or consists of the amino acid sequence of one of SEQ ID NOs: 1, 11, 21, 31, and 41.

9. The method of any one of claims 1-8, wherein the autoimmune disease is selected from the group consisting of inflammatory bowel disease (IBD), multiple sclerosis, rheumatoid arthritis, psoriasis, diabetes, systemic lupus erythematosus (SLE), and ankylosing spondylitis (AS).

10. The method of any one of claims 1-8, wherein the atopic disease is selected from allergic asthma, atopic dermatitis, allergic rhinitis, food allergy, and allergic conjunctivitis.

11. A method of treating cancer in a subject in need thereof, comprising administering an anti-CDld antibody to the subject, wherein the anti-CDld antibody binds over the A’ pocket of CD Id expressed on a target cell, and wherein the anti-CDld antibody does not induce cell death of the target cell and administering a second antibody that binds to phosphatidylserine in complex with beta2 glycoprotein 1 (B2GP1) to the subject.

12. The method of claim 11, wherein binding of the antibody increased CD Id presentation of phosphatidylserine on the target cell13. The method of claim 11 or claim 12, wherein the anti-CDld antibody increases T-cell immunoglobulin and mucin domain (TIM)-1, TIM-3, and / or TIM-4 binding to the target cell.

14. The method of any one of claims 11-13, wherein the anti-CDld antibody modulates TIM-I, TIM-3, and / or TIM-4 signaling.

15. The method of any one of claims 11-14, wherein the anti-CDld antibody is a single domain antibody.

16. The method of claim 15, wherein the single domain antibody comprises:(a) a CDR1 sequence selected from SEQ ID NO:2, 5, and 8, a CDR2 sequence selected from SEQ ID NO: 3, 6, and 9, and a CDR3 sequence selected from SEQ ID NO: 4, 7 and 10;(b) a CDR1 sequence selected from SEQ ID NO: 12, 15, and 18, a CDR2 sequence selected from SEQ ID NO: 13, 16, and 19, and a CDR3 sequence selected from SEQ ID NO: 14,17. and 20;(c) a CDR1 sequence selected from SEQ ID NO:22, 25, and 28, a CDR2 sequence selected from SEQ ID NO: 23, 26, and 29, and a CDR3 sequence selected from SEQ ID NO: 24, 27, and 30;(d) a CDR1 sequence selected from SEQ ID NO:32, 35, and 38, a CDR2 sequence selected from SEQ ID NO: 33, 36, and 39, and a CDR3 sequence selected from SEQ ID NO: 34, 37, and 40; or(e) a CDR1 sequence selected from SEQ ID NO:42, 45 and 48, a CDR2 sequence selected from SEQ ID NO: 43, 46, and 49, and a CDR3 sequence selected from SEQ ID NO: 44, 47, and 50.

17. The method of claim 16, wherein the single domain antibody comprises an amino acid sequence that it at least 90%, at least 95%, or at least 99% identical to one of SEQ ID NOs: 1,I I, 21, 31, or 41.

18. The method of claim 16 or claim 17, wherein the single domain antibody comprises or consists of the amino acid sequence of one of SEQ ID NOs: 1, 11, 21, 31, or 41.

19. The method of any one of claims 11-18, wherein the second antibody is bavituximab.

20. The method of any one of claims 11-19, wherein the anti-CDld antibody is administered prior to the second antibody.

21. The method of any one of claims 11-19, wherein the anti-CDld antibody and the second antibody are administered simultaneously.

22. The method of any one of claims 11-21, wherein the cancer expresses CD Id.

23. The method of any one of claims 11-22, wherein the cancer is selected from a myeloid malignancy, a B-cell malignancy, renal cell carcinoma, medulloblastoma, and glioma.

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