CD48 variants and fusion proteins thereof

Abstract

Provided are CD48 fragments and Fc fusion proteins thereof that include mutations to increase binding affinities to CD48 receptors, such as CD2 and / or 2B4. Also provided are pharmaceutical compositions comprising the CD48 fragments and Fc fusion proteins thereof, methods of producing these proteins, methods of using these proteins to reduce lymphocyte activity, and methods of using these proteins to treat autoimmune and inflammatory diseases.

Description

Attorney Docket No: IMID-102WOCD48 VARIANTS AND FUSION PROTEINS THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 746,006, filed on January 16, 2025, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 6, 2025, is named IMID-102WO_SL.xml and is 51,008 bytes in size.FIELD OF THE DISCLOSURE

[0003] The disclosure relates to CD48 fragments and Fc fusion proteins thereof that include mutations to increase binding affinities to CD48 receptors, such as CD2 and / or 2B4. The disclosure also relates to methods of making these proteins, methods of using these proteins to reduce lymphocyte activity, and methods of using these proteins to treat autoimmune and inflammatory diseases.BACKGROUND

[0004] Cluster of Differentiation 48 (CD48), also known as B-lymphocyte activation marker (BLAST-1) or signaling lymphocytic activation molecule 2 (SLAMF2), is a member of the CD2 subfamily of the immunoglobulin superfamily which includes SLAM (signaling lymphocyte activation molecules) proteins. CD48 is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein found on the surface of lymphocytes and other immune cells such as dendritic cells. CD48 includes a membrane-distal immunoglobulin variable-like (IgV) domain and a membrane-proximal immunoglobulin constant-2 (IgC2) domain.

[0005] CD48 interacts with 2B4 (also known as CD244 or SLAMF4), a receptor expressed on natural killer (NK) cells, a subset of cytotoxic T cells, monocytes, basophils, dendritic cells, and myeloid-derived suppressor cells. Binding of CD48 to 2B4 can transmit either an activating or an inhibitory signal. The expression levels of 2B4 and the presence of adaptor molecules, which bind to its cytoplasmic tail, such as intracellular SLAM-associated protein (SAP) or Ewing sarcoma-activated transcript 2 (EAT2) determine whether 2B4Attorney Docket No: IMID-102WObinding propagates an activating or an inhibitory signal. 2B4 transmits activating signals in cells expressing abundant amounts of SAP, such as cytotoxic T and NK cells (Agresta et al., FRONT. IMMUNOL. (2018) 9:2809).

[0006] CD48 also weakly binds CD2 in humans. In mice, CD48 is a high affinity ligand of CD2. CD2 is a co-stimulatory receptor expressed on NK cells and T cells. Its interaction with lymphocyte-function antigen 3 (LFA3 also known as CD58), a cell surface protein expressed on antigen-presenting cells, facilitate the formation and organization of the immunological synapse between T cells and antigen-presenting cells (Binder et al.. FRONT. IMMUNOL. (2020) 11:1090). CD2 expression is upregulated on activated T cells and memory T cells, and plays an important role in activation of memory T cells despite the coexistence of several other costimulatory pathways.

[0007] CD48 and its receptors are implicated in multiple immune cell functions. There remains a need to develop therapeutic proteins that can modulate their interactions, for treating associated diseases and disorders.SUMMARY OF THE INVENTION

[0008] The disclosure is based, in part, on the identification of mutations in CD48 (e.g., human CD48) immunoglobulin variable-like (IgV) domain that increase binding affinities to 2B4 (e.g., human 2B4) and / or CD2 (e.g., human CD2). CD48 IgV domains incorporating these mutations can be incorporated in soluble protein constructs that compete with endogenous ligands, such as membrane-attached CD48, for binding 2B4 and / or CD2, thereby to reduce 2B4- and / or CD2-mediated immune cell (e.g., NK cell and T cell) activation. Such soluble protein constructs can include an IgG Fc region for dimerization and serum half-life extension. The soluble protein constructs are useful for treating autoimmune and inflammatory diseases.

[0009] Accordingly, in one aspect, the present disclosure provides a CD48 IgV domain variant comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1 and / or at least 80% identical to SEQ ID NO: 15, the CD48 IgV domain variant comprising one or more mutations at one or more amino acid positions selected from 27, 34, 43-46, 76-78, 86, 88, and 89, corresponding to SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant does not contain just a single point mutation of a Tyr (Y) for Gin (Q) at amino acid positionAttorney Docket No: IMID-102WO

[0010] The CD48 IgV domain variant can include one or more of: Glu (E) at amino acid position 27, Tyr (Y) at amino acid position 34, Vai (V) at amino acid position 43, Lys (K) at amino acid position 44, Arg (R) at amino acid position 45, Leu (L) at amino acid position 46, Ala (A) at amino acid position 76, Asn (N) at amino acid position 77, He (I) at amino acid position 78, Asp (D) at amino acid position 86, Met (M) at amino acid position 88, and Phe (F) at amino acid position 89, corresponding to SEQ ID NO: 1. Specifically, the CD48 IgV domain variant can include one or more of:(a) Glu (E) at amino acid position 27 ;(b) Tyr (Y) at amino acid position 34;(c) Vai (V) at amino acid position 43, Lys (K) at amino acid position 44, Arg (R) at amino acid position 45, and Leu (L) at amino acid position 46;(d) Ala (A) at amino acid position 76, Asn (N) at amino acid position 77, and He (I) at amino acid position 78;(e) Asp (D) at amino acid position 86; and(f) Met (M) at amino acid position 88, and Phe (F) at amino acid position 89.One example of the CD48 IgV domain variant includes the mutation of (a) above. Another example of the CD48 IgV domain variant includes the mutations of (d) above.

[0011] In certain embodiments, the CD48 IgV domain variant includes the mutations of (a), (d), and (f) above. Such CD48 IgV domain variant can include an amino acid sequence at least 90% identical to SEQ ID NO: 11. In a specific example, the CD48 IgV domain variant includes the amino acid sequence of SEQ ID NO: 11.

[0012] In certain embodiments, the CD48 IgV domain variant includes the mutations of (a), (b), (c), (d), and (f) above. Such CD48 IgV domain variant can include an amino acid sequence at least 90% identical to SEQ ID NO: 38. In a specific example, the CD48 IgV domain variant includes the amino acid sequence of SEQ ID NO: 38.

[0013] In certain embodiments, the CD48 IgV domain variant includes the mutations of (a), (b), (d), and (f) above. Such CD48 IgV domain variant can include an amino acid sequence at least 90% identical to SEQ ID NO: 12. In a specific example, the CD48 IgV domain variant includes the amino acid sequence of SEQ ID NO: 12.

[0014] In certain embodiments, the CD48 IgV domain variant includes the mutations of (a), (d), (e) and (f) above. Such CD48 IgV domain variant can include an amino acidAttorney Docket No: IMID-102WOsequence at least 90% identical to SEQ ID NO: 13. In a specific example, the CD48 IgV domain variant includes the amino acid sequence of SEQ ID NO: 13.

[0015] In certain embodiments, the CD48 IgV domain variant includes the mutations of (a), (b), (d), (e) and (f) above. Such CD48 IgV domain variant can include an amino acid sequence at least 90% identical to SEQ ID NO: 14. In a specific example, the CD48 IgV domain variant includes the amino acid sequence of SEQ ID NO: 14.

[0016] In certain embodiments, the CD48 IgV domain variant includes the mutations of (a), (b), (c), (d), (e) and (f) above. Such CD48 IgV domain variant can include an amino acid sequence at least 90% identical to SEQ ID NO: 15. In a specific example, the CD48 IgV domain variant includes the amino acid sequence of SEQ ID NO: 15.

[0017] In certain embodiments, the CD48 IgV domain variant includes the mutations of (a), (c), (d), and (f) above.

[0018] The CD48 IgV domain variants disclosed herein can comprise an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1; and / or have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to similarity to SEQ ID NO: 1. The CD48 IgV domain variants can comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 amino acid mutations relative to SEQ ID NO: 1.

[0019] The CD48 IgV domain variants disclosed herein can comprise an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15; and / or have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to SEQ ID NO: 15.

[0020] The CD48 IgV domain variants can have increased binding affinity to CD2 and / or 2B4. Eor example, a CD48 IgV domain variant disclosed herein can bind CD2 at a higher affinity than a CD48 IgV domain comprising the amino acid sequence of SEQ ID NO: 1, and / or at a higher affinity than a corresponding CD48 IgV domain that does not include the one or more mutations. In another example, a CD48 IgV domain variant disclosed herein can bind 2B4 at a higher affinity than a CD48 IgV domain comprising the amino acid sequence of SEQ ID NO: 1, and / or at a higher affinity than a corresponding CD48 IgV domain that does not include the one or more mutations.Attorney Docket No: IMID-102WO

[0021] In another aspect, the present disclosure provides a CD48 fragment comprising a CD48 IgV domain variant disclosed herein. The CD48 fragment can further include a CD48 IgC2 domain, for example, a CD48 IgC2 domain comprising an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 17. A C-terminus of the CD48 IgV domain can be linked, directly or indirectly, to an N-terminus of the CD48 IgC2 domain. Where the linkage is indirect, the C-terminus of the CD48 IgV domain can be linked to the N-terminus of the CD48 IgC2 domain via a peptide linker comprising the amino acid sequence of SEQ ID NO: 18.

[0022] In another aspect, the present disclosure provides a CD48-Fc fusion polypeptide comprising (i) a CD48 fragment disclosed herein; and (ii) an IgG Fc region. The IgG Fc region can be an IgGl, IgG2, IgG3, or IgG4 Fc region. Specifically, the IgG Fc region can be a human IgGl Fc region, which optionally includes one or more mutations that reduce antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) effector functions, for example, both ADCC and CDC effector functions. Such mutations include but are not limited to one or more mutations at positions 234, 235, 237, 329, 330, and / or 331, according to the EU numbering system, for example, one or more mutations selected from L234A, L235A, L235E, G237A, P329A, A330S, and P331S. A specific set of mutations contemplated is L234A and L235A substitutions in the human IgGl Fc region. The IgG Fc region fused to the CD48 fragment can also be a human IgG4 Fc region. In the CD48-Fc fusion polypeptide a C-terminus of the CD48 fragment is linked, directly or indirectly, to an N-terminus of the IgG Fc region. For example, the C-terminus of the CD48 fragment can be linked to the N-terminus of the IgG Fc region via a peptide linker, such as a peptide linker comprising the amino acid sequence of SEQ ID NO: 9.

[0023] In another aspect, the present disclosure provides a CD48-Fc fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 39. In another aspect, the present disclosure provides a CD48-Fc fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 40.

[0024] In another aspect, the present disclosure provides a CD48-Fc fusion protein comprising two CD48-Fc fusion polypeptides disclosed herein, wherein the two CD48-Fc fusion polypeptides form a dimer (e.g., homodimer). The two CD48-Fc fusion polypeptides can be linked by one or more covalent bonds, such as one or more disulfide bonds. In certain embodiments, the CD48-Fc fusion protein is a soluble protein.Attorney Docket No: IMID-102WO

[0025] In another aspect, the present disclosure provides a pharmaceutical composition comprising: a CD48 IgV domain variant, a CD48 fragment, a CD48-Fc fusion polypeptide, or a CD48-Fc fusion protein disclosed herein, and a pharmaceutically acceptable carrier.

[0026] In another aspect, the present disclosure provides an isolated nucleic acid encoding a CD48 IgV domain variant, a CD48 fragment, or a CD48-Fc fusion polypeptide disclosed herein. Also provided are a vector comprising the nucleic acid and a recombinant cell comprising the nucleic acid or the vector. The present disclosure further provides a method of producing a protein, the method comprising culturing the recombinant cell under conditions to allow expression of the CD48 IgV domain variant, the CD48 fragment, or the CD48-Fc fusion polypeptide encoded by the nucleic acid. The method can further include purifying the CD48 IgV domain variant, the CD48 fragment, or the CD48-Fc fusion polypeptide, optionally followed by an additional step of formulating the CD48 IgV domain variant, the CD48 fragment, or the CD48-Fc fusion polypeptide with a pharmaceutically acceptable carrier.

[0027] In another aspect, the present disclosure provides a method of inhibiting the activity of a NK cell or a CD8+T cell, the method comprising contacting the NK cell or the CD8+T cell with a CD48 IgV domain variant, a CD48 fragment, a CD48-Fc fusion polypeptide, or a CD48-Fc fusion protein disclosed herein.

[0028] In another aspect, the present disclosure provides a method of treating an autoimmune disease, the method comprising administering to a subject in need thereof an effective amount of a CD48 IgV domain variant, a CD48 fragment, a CD48-Fc fusion polypeptide, a CD48-Fc fusion protein, or a pharmaceutical composition disclosed herein, thereby to treat the autoimmune disease. The present disclosure also provides a method of treating an inflammatory disease, the method comprising administering to a subject in need thereof an effective amount of a CD48 IgV domain variant, a CD48 fragment, a CD48-Fc fusion polypeptide, a CD48-Fc fusion protein, or a pharmaceutical composition disclosed herein, thereby to treat the inflammatory disease. In these methods, the CD48 IgV domain variant, the CD48 fragment, the CD48-Fc fusion polypeptide, the CD48-Fc fusion protein, or the pharmaceutical composition can be administered to the subject intravenously or subcutaneously.

[0029] Other embodiments and details of the disclosure are presented herein below.Attorney Docket No: IMID-102WOBRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a schematic illustrating an exemplary CD48-Fc fusion protein. The fusion protein is a homodimer with two polypeptide chains, each including, from N-terminus to C-terminus, a CD48 IgV domain, a CD48 IgC2 domain, and an IgG Fc region including a hinge, a CH2 domain, and a CH3 domain.

[0031] FIGs. 2A-2C show the binding of wild-type hCD48-Fc, A43-Fc and hCD48-Fc variants containing single residue mutations in the IgV domain, to human YT cells, which endogenously express h2B4, and Jurkat cells that endogenously express hCD2. FIG. 2A is a series of histograms showing representative cytometry results of cells incubated with wildtype hCD48-Fc, A43-Fc, hCD58-Fc (Jurkat cells only), and an unrelated control Fc fusion protein (Ctl-Fc) (grey peaks), and without any Fc fusion proteins (dashed line unfilled peaks).FIG. 2B is a series of histograms showing representative cytometry results of cells incubated with hCD48-Fc SI, hCD48-Fc S2, hCD48-Fc S3, and hCD48-Fc S4 domain variants (grey peaks), and without any Fc fusion proteins (dashed line unfilled peaks). FIG. 2C is a series of bar graphs showing the mean fluorescence intensity (MFI) of soluble Fc fusion protein interactions with YT or Jurkat cells. Data are represented as the mean ± standard deviation of duplicate samples.

[0032] FIGs. 3A and 3B show the binding of hCD48-Fc variants containing individual mutations in adjacent residues of the IgV domain, to human YT cells, which endogenously express h2B4, and Jurkat cells that endogenously express hCD2. FIG. 3A is a series of histograms showing representative cytometry results of cells incubated with hCD48-Fc Al, hCD48-Fc A2, or hCD48-Fc A3 (grey peaks), and without any Fc fusion proteins (dashed line unfilled peaks). FIG. 3B is a series of bar graphs showing the MFI of soluble Fc fusion protein interactions with YT or Jurkat cells. Data are represented as the mean ± standard deviation of duplicate samples.

[0033] FIGs. 4A and 4B show the binding of hCD48-Fc variants, containing a combination of mutations in single and adjacent residues of the IgV domain, to human YT cells that endogenously express h2B4, and Jurkat cells that endogenously express hCD2. FIG. 4A is a series of histograms showing representative cytometry results of cells incubated with hCD48-Fc Cl, hCD48-Fc C2, hCD48-Fc C3, hCD48-Fc C4, hCD48-Fc C5 (grey peaks), and without any Fc fusion proteins (dashed line unfilled peaks). FIG. 4B is a series of bar graphs showing the MFI of soluble protein interactions with YT or Jurkat cells. Data are represented as the mean ± standard deviation of duplicate samples.Attorney Docket No: IMID-102WO

[0034] FIGs. 5A and 5B show the interaction of hCD48-Fc SI, hCD48-Fc Cl, hCD48-Fc C4, and hCD48-Fc C5 variants with COS-7 cells transiently transfected with a h2B4-expressing vector (also referred to as “COS-7 h2B4 cells” below) or with a hCD2-expressing vector (also referred to as “COS-7 hCD2 cells” below). FIG.5A is a series of histograms showing representative cytometry results of COS-7 cells (upper panels), COS-7 h2B4 cells (intermediate panels), or COS-7 hCD2 cells (lower panels) incubated with purified wild-type hCD48-Fc, hCD58-Fc, hCD48-Fc SI, hCD48-Fc Cl, hCD48-Fc C4, hCD48-Fc C5, or an unrelated control Fc fusion protein (Ctl-Fc) (filled picks), and without Fc fusion proteins (dashed line unfilled peaks). FIG. 5B is a bar graph showing the MFI of soluble Fc fusion protein interactions with COS-7, COS-7 h2B4, and COS-7 hCD2 cells. Data are represented as the mean ± standard deviation of duplicate samples.

[0035] FIGs. 6A and 6B are graphs showing dose-response curves of the binding of purified hCD48-Fc SI , hCD48-Fc Cl, hCD48-Fc C4 and hCD48-Fc C5 variants, wild-type hCD48-Fc, or an unrelated control Fc fusion protein (Ctl-Fc) to human YT cells that endogenously express h2B4 (FIG. 6A), or Jurkat cells that endogenously express hCD2 (FIG. 6B) analyzed by flow cytometry. The plots illustrate MFI data resulting from the binding of the soluble Fc fusion proteins to YT or Jurkat cells. Each data point is represented as the mean ± standard deviation of duplicate samples.

[0036] FIGs. 7A and 7B are graphs showing dose-response curves of the binding of purified hCD48-Fc SI, hCD48-Fc Cl, hCD48-Fc C4 and hCD48-Fc C5 variants, wild-type hCD48-Fc, hCD58-Fc (to hCD2 only), or an unrelated Fc control protein (Ctl-Fc) to h2B4 (FIG. 7A) or hCD2 (FIG.7B) analyzed by ELISA. The plots illustrate OD data resulting from the binding of the soluble Fc fusion proteins to h2B4 or hCD2 immobilized on the plates. Each data point is represented as the mean ± standard deviation of duplicate samples.

[0037] FIGs. 8A and 8B are bar graphs showing the blockage of the interactions between hCD48 and h2B4 (FIG. 8A) and hCD58 and hCD2 (FIG. 8B) by different concentrations of purified hCD48-Fc Cl, hCD48-Fc C4 and hCD48-Fc C5 variants, wild-type hCD48-Fc, wildtype hCD58-Fc, or an unrelated control Fc fusion protein, analyzed by ELISA. The percentages of blocking for each Fc fusion protein were calculated from the OD values obtained in the ELISA, considering as 100% interaction the OD values in the absence of competing soluble Fc fusion protein. FIGs. 8C and 8D are bar graphs showing competitive inhibition of hCD48:h2B4 and hCD58:hCD2 interactions assessed by flow cytometry. HEK cells transfected with h2B4 (FIG. 8C) and Jurkat cells (FIG. 8D) were incubated withAttorney Docket No: IMID-102WOvarying concentrations of purified hCD48-Fc, hCD48-Fc C5, hCD58-Fc, or an unrelated control Fc protein (Ctl-Fc), followed by CD48-mFc (for HEK h2B4 cells) or CD58-mFc (for Jurkat cells) and analyzed by flow cytometry using an anti-mouse IgG Fc-PE. The percentage of interaction blockade was derived from the measured mean fluorescence intensity (MFI) values. Data are represented as the mean ± standard deviation of triplicate samples.

[0038] FIGs. 9A-9C are bar graphs showing the inhibitory effect of hCD48-Fc Cl, hCD48-Fc C4, and hCD48-Fc C5 variants on the formation of cell conjugates mediated by hCD48 and h2B4 or hCD58 and hCD2. In FIG. 9A CMAC-labeled YT cells, which endogenously express h2B4, were incubated with CFSE-labeled HEK cells transfected with hCD48, in the absence or presence of purified wild-type hCD48-Fc, hCD48-Fc Cl, hCD48-Fc C4, or hCD48-Fc C5 proteins, or an unrelated Fc control protein (Ctl-Fc). In FIG. 9B CMAC-labeled Jurkat cells, endogenously expressing hCD2, were incubated with CFSE-labeled K562 cells, which endogenously express hCD58, in the absence or presence of wildtype hCD48-Fc*, hCD58-Fc*, hCD48-Fc* Cl, hCD48-Fc* C4, or hCD48-Fc* C5 proteins, or an unrelated Fc control protein (Ctl-Fc*). In FIG. 9C CMAC-labeled YT cells, which endogenously express h2B4, were incubated with CFSE-labeled K562 cells transfected with hCD48, in the absence or presence of purified wild-type hCD48-Fc#, or hCD48-Fc#C5 proteins, or an unrelated Fc control protein (Ctl-Fc). The graphs represent the percentages of blockage of conjugates (detected as % of double positive events) formed in the samples analyzed after 20 min of incubation at 37°C. The percentages of blockage for each Fc fusion protein were calculated considering as 100% the formation of conjugate values obtained in the absence of soluble Fc fusion protein. The mean and standard deviation of two (FIG. 9A and 9B) or three (FIG. 9C) independent samples is represented. The Fc of the Fc fusions proteins used in the experiments involving Jurkat and K562 cells were human IgGl Fc regions with mutations L234F, L235Q, K322Q, M252Y, S254T, and T256E, which abrogate the binding of the Fc region to Fc receptors (Fc*). Another Fc variant used in the experiments included mutations L234A, L235A, M252Y, S254T, and T256E, and the corresponding fusion proteins were indicated as Fc#proteins.

[0039] FIG. 10A is a bar graph showing the inhibitory effect of the purified hCD48-Fc C5 variant on NK-cell cytotoxicity mediated by hCD48 and h2B4 interactions. YT cells, which endogenously express h2B4, were pre-incubated with wild-type hCD48-Fc, hCD48-Fc C5, or an unrelated Fc control protein (Ctl-Fc), or incubated without Fc fusion protein (Ctl), and subsequently exposed to calcein-labeled HEK cells or hCD48 transfected HEK cells atAttorney Docket No: IMID-102WOeffector cell / target cell (E / T) ratios of 5 / 1, or 10 / 1, for 4 h at 37°C. NK-cell cytotoxicity, depicted as %, is calculated according to the formula: (experimental release — spontaneous release) / (maximum releases — spontaneous release) x 100. The mean and standard deviation of quintuplicate samples is represented. FIG. 10B is a bar graph showing the inhibitory effect of the purified hCD48-Fc#C5 variant on NK-cell cytotoxicity. YT cells were pre-incubated with 5 pg / mL of purified hCD48-Fc#, hCD48-Fc#C5, or an unrelated control Fc" protein (Ctl-Fc#), or untreated (Ctl+) and subsequently exposed to calcein-labeled K562 cells or hCD48-stably transfected K562 cells at effector cell / target cell (E / T) ratios of 2 / 1, or 5 / 1, for 4 h at 37°C. The fluorescence emitted by the calcein released to the supernatant was measured and the percentage of cytotoxicity of YT cells against target cells calculated. The mean and standard deviation of triplicate samples are presented.

[0040] FIG. 11 is a graph showing the inhibitory effect of the purified hCD48-Fc#C5 variant on NK-cell cytotoxicity mediated by hCD58 and hCD2 interactions. NK-92 cells, which endogenously express hCD2, were pre-incubated with wild-type hCD48-Fc#, hCD58-Fc#, hCD48-Fc#C5, or an unrelated Fc#control protein (Ctl-Fc#), or incubated without any Fc fusion protein (Ctl), and subsequently exposed to calcein-labeled K562 cells, which endogenously express hCD58, at effector cell / target cell (E / T) ratios of 0.5 / 1, or 2 / 1, for 4 h at 37°C. NK-cell cytotoxicity, depicted as %, is calculated according to the formula:(experimental release — spontaneous release) / (maximum release — spontaneous release) x 100. The mean and standard deviation of quintuplicate samples is represented.

[0041] FIGs. 12A-12E are graphs showing the inhibitory effect of the purified hCD48-Fc" C5 variant on T-cell proliferation and cytokine production in allogeneic mixed lymphocyte reaction (MLR) assays. CFSE-labeled responder PBMCs (PBLs) were stimulated with irradiated allogeneic PBMCs (PBLs) at a 1:1 ratio for 6 days in the absence or presence of 5 pg / mL of purified hCD48-Fc#, hCD48-Fc#C5, hCD58-Fc#, or an unrelated control Fc#protein (Ctl-Fc#). (FIG. 12A-12C) Proliferation of total T cells (FIG. 12A), CD4+T cells (FIG. 12B), and CD8+T cells (FIG. 12C) was quantified from 7 donor pairs by measuring CFSE dilution via flow cytometry. Supernatants from 8 donor pairs were analyzed for IFN-y (FIG. 12D) and TNF-a (FIG. 12E) production by ELISA. Values were normalized to cocultures without Fc#protein. Each symbol represents one donor pair; horizontal bars indicate mean values.

[0042] FIG. 13 is a plot showing both the non-adjusted and self-adjusted risk scores along the sequence of hCD48-Fc"-C5.Attorney Docket No: IMID-102WO

[0043] FIGs. 14A and 14B show the interaction of hCD48-Fc#C5 and derivative variants with YT cells and Jurkat cells. FIG. 14A is a bar graph showing the MFI of soluble Fc" fusion protein interactions with YT cells, which endogenously express hCD2, incubated with increasing concentrations of purified hCD48-Fc#C5, hCD48-Fc#mH, hCD48-Fc#mI, hCD48-Fc#Rl, or an unrelated control Fc#protein (Ctl-Fc#) as indicated, followed by an antihuman IgG Fc-PE antibody, and analyzed by flow cytometry. FIG. 14B is a bar graph showing the MFI of soluble Fc#fusion protein interactions with Jurkat cells, which endogenously express hCD2, incubated with increasing concentrations of purified hCD48-Fc#C5, hCD48-Fc#mH, hCD48-Fc#mI, hCD48-Fc#Rl, or an unrelated control Fc#protein (Ctl-Fc#) as indicated, followed by an anti-human IgG Fc-PE antibody, and analyzed by flow cytometry. Data are presented as the mean ± standard deviation of duplicate samples.

[0044] FIGs. 15A and 15B are graphs showing the inhibitory effect of hCD48-Fc#C5 and derivative variants on T-cell proliferation and cytokine production in allogeneic mixed lymphocyte reaction (MLR) assays. CFSE-labeled responder PBLs were stimulated with irradiated allogeneic PBLs at a 1 / 1 ratio for 6 days in the absence or presence of 5 Lig / m of purified hCD48-Fc#C5, hCD48-Fc#mH, hCD48-Fc#ml, hCD48-Fc#R1 , or an unrelated control Fc#protein (Ctl-Fc#). Control cultures included irradiated PBLs incubated with autologous CFSE-labeled PBLs. FIG. 15A shows proliferation of T cells quantified at day 6 of co-culture from 6 donor pairs by measuring CFSE dilution via flow cytometry. Values were normalized to co-cultures without Fc# protein. Percentage of inhibition was determined based on the reduction of proliferation levels in co-cultures incubated with the protein of interest, compared to control co-cultures without any Fc#protein. Data are presented as bar plots showing the mean ± standard deviation of the various responder-stimulator pairs. FIG.15B shows IFN-y production analyzed from supernatants of 10 donor pairs by ELISA. Values were normalized to co-cultures without Fc#protein. Percentage of inhibition was determined based on the reduction of IFN-y levels in co-cultures incubated with the protein of interest, compared to control co-cultures without any protein. Each symbol corresponds to a single responder-stimulator pair and horizontal bars indicate the mean values.DETAILED DESCRIPTION

[0045] The disclosure is based, in part, on the identification of mutations in CD48 (e.g., human CD48) immunoglobulin variable-like (IgV) domain that increase binding affinities to 2B4 (e.g., human 2B4) and / or CD2 (e.g., human CD2). CD48 IgV domains incorporating these mutations can be incorporated in soluble protein constructs that compete withAttorney Docket No: IMID-102WOendogenous ligands, such as membrane-attached CD48, for binding 2B4 and / or CD2, thereby to reduce 2B4- and / or CD2-mediated immune cell (e.g., NK cell and T cell) activation. Such soluble protein constructs can include an IgG Fc region for dimerization and serum half-life extension. The soluble protein constructs are useful for treating autoimmune and inflammatory diseases.1. Definitions

[0046] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0047] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0048] As used herein, all the amino acid positions in an Fc region are numbered according to EU numbering.

[0049] The terms “polypeptide,” “protein,” and “peptide” refer to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation).

[0050] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences e.g., two polypeptides or two nucleic acids have the same respective amino acid or nucleotide at the same positions in an alignment. As used herein, “percent identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Similarly, “percent identity” between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST- 2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) NATURE GENETICS 6: 119-129, which is fully incorporated by reference herein. ThoseAttorney Docket No: IMID-102WOskilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0051] The alignment algorithms above may take into account a scoring matrix to calculate an alignment score (see Chao et al., BIOMOT.ECUI.ES (2022) 12(4): 546). For example, for an amino acid sequence at least 85 amino acids in length, the scoring matrix recommended by the BLAST algorithm is BLOSUM-62. The BLOSUM-62 scoring matrix assigns positive, zero, or negative scores between each pair or standard amino acid residues (see Henikoff and Henikoff, PROC. NATL. ACAD. Set. USA (1992) 89, 10915-19 at FIG. 2). A positive score between two amino acid residues indicates that substitution of these amino acid residues for each other is conservative. As used herein, “similarity” between a subject amino acid sequence and a reference amino acid sequence refers to the percentage of amino acid residues in the subject amino acid sequence that are identical or have a conservative substitution according to the BLOSUM-62 scoring matrix, relative to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence alignment score.

[0052] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.

[0053] As used herein, the term “effective amount” refers to the amount of an active agent sufficient to effect beneficial or desired results (e.g., a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administration(s), application! s) or dosage(s) and is not intended to be limited to a particular formulation or administration route.

[0054] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0055] The terms “treat,” “treating,” or “treatment,” and other grammatical equivalents as used in this disclosure, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arrestingAttorney Docket No: IMID-102WOthe development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. The term “therapeutic benefit” refers to eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.

[0056] As used herein, the tern “combination” in the context of therapies means that two or more different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0057] The term “about” refers to any minimal alteration to a given value, including ±5%, ±10%, or ±15% of a specified numerical value or data point. Ranges can be expressed in this disclosure as from “about” one particular value, and / or to “about” another particular value. When values are expressed as approximations by use of the antecedent “about,” it is understood that the disclosure also contemplates embodiments that specify the particular values and ranges of values without the approximations. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of valuesAttorney Docket No: IMID-102WOdisclosed in this disclosure, and that each value is also disclosed as “about” that particular value in addition to the value itself.

[0058] Throughout the description, where compositions are described as having, including, containing, incorporating, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is intended that compositions and methods are inclusive or open-ended and do not exclude additional, unrecited components or steps. It is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps.

[0059] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0060] It should be understood that the expression “at least one of’ or “one or more of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0061] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.Attorney Docket No: IMID-102WO

[0062] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.IL CD48 Variants and Fusion Proteins

[0063] The present disclosure provides CD48 IgV domain variants and fusion proteins comprising the variants.A. CD48 IgV Domain Variants

[0064] The IgV domain of a wild-type, mature human CD48 has an amino acid sequence set forth in SEQ ID NO: 1. In one aspect, a CD48 IgV domain variant of the present disclosure has increased binding affinities to CD2 and / or 2B4 relative to a wild-type CD48 IgV domain (e.g., a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1). A CD48 IgV domain variant can comprise at least one mutation (e.g., a substitution, deletion, insertion, or modification of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) relative to the wild-type CD48 IgV domain. In certain embodiments, such a mutation results in a CD48 IgV domain variant that has an increased binding affinity to CD2 (e.g., human CD2). In certain embodiments, such a mutation results in a CD48 IgV domain variant that has an increased binding affinity to 2B4 (e.g., human 2B4). In certain embodiments, such a mutation results in a CD48 IgV domain variant that has increased binding affinities to both 2B4 (e.g., human 2B4) and CD2 (e.g., human CD2).

[0065] The amino acid sequences of a wild-type human CD48 IgV domain and exemplary variants are provided in Table 1.Table 1: CD48 IgV variant sequencesAttorney Docket No: IMID-102WOAttorney Docket No: IMID-102WO*The amino acid residues shown in bold and underlined are amino acid substitutions in the human CD48 IgV background that increase binding affinity to 2B4 and / or CD2.

[0066] In some embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 65%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the wild-type human CD48 amino acid sequence of SEQ ID NO: 1. In some embodiments, the CD48 IgV domain variant comprises an amino acid sequence no greater than 99%, no greater than 98%, no greater than 97%, no greater than 96%, no greater than 95%, no greater than 94%, no greater than 93%, no greater than 92%, no greater than 91%, or no greater than 90% identical to SEQ ID NO: 1. In some embodiments, the CD48 IgV domain variant comprises an amino acid sequence having at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to SEQ ID NO: 1. In some embodiments, the CD48 IgV domain variant comprises an amino acid sequence having no greater than 99%, no greater than 98%, no greater than 97%, no greater than 96%, no greater than 95%, no greater than 94%, no greater than 93%, no greater than 92%, no greater than 91%, or no greater than 90% similarity to SEQ ID NO: 1. In some embodiments, the CD48 IgV domain variant comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 amino acid mutations relative to SEQ ID NO: 1.

[0067] In some embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15. In some embodiments, the CD48 IgV domain variantAttorney Docket No: IMID-102WOcomprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to SEQ ID NO: 15.

[0068] Where the CD48 IgV domain variant aligns only to a fragment of SEQ ID NO: 1, it is also contemplated that in some embodiments, the fragment constitutes at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the fragment is a single contiguous portion of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the fragment comprises two or more portions of the amino acid sequence of SEQ ID NO: 1, wherein the two or more portions are not contiguous with each other. In some embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the fragment of SEQ ID NO: 1. In some embodiments, the CD48 IgV domain variant comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 amino acid mutations relative to the fragment of SEQ ID NO: 1. In some embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a fragment of SEQ ID NO: 15 that corresponds to the fragment of SEQ ID NO: 1.

[0069] In certain embodiments, the CD48 IgV domain variant comprises one or more amino acid mutations (e.g., substitutions) in amino acid positions 27, 34, 43, 44, 45, 46, 76, 77, 78, 86, 88, and / or 89, numbered according to SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, a substitution of the phenylalanine (F) at position 34, a substitution of the serine (S) at position 43, a substitution of the arginine (R) at position 44, a substitution of the lysine (K) at position 45, a substitution of the serine (S) at position 46, a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, a substitution of the threonine (T) at position 78, a substitution of the lysine (K) at position 86, a substitution of the glycine (G)Attorney Docket No: IMID-102WOat position 88, and / or a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1.

[0070] In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E), the substitution of the phenylalanine (F) at position 34 is to a tyrosine (Y), the substitution of the serine (S) at position 43 is to a valine (V), the substitution of the arginine (R) at position 44 is to a lysine (K), the substitution of the lysine (K) at position 45 is to an arginine (R), the substitution of the serine (S) at position 46 is to a leucine (L), the substitution of the asparagine (N) at position 76 is to an alanine (A), the substitution of the serine (S) at position 77 is to an asparagine (N), the substitution of the threonine (T) at position 78 is to an isoleucine (I), the substitution of the lysine (K) at position 86 is to an aspartic acid (D), the substitution of the glycine (G) at position 88 is to a methionine (M), and / or the substitution of the asparagine (N) at position 89 is to a phenylalanine (F).

[0071] In certain embodiments, the CD48 IgV domain variant binds at least one of CD2 and 2B4, or an extracellular fragment thereof, at an increased binding affinity compared to a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1. The affinity can be measured by methods known in the art, such as surface plasmon resonance or flow cytometry to determine the amount of CD48 IgV domain variants, in a monomeric or dimeric (e.g., Fc fused) form, bound to CD2- or 2B4-expressing cells as indicated by MFI.

[0072] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a glutamic acid (E) at position 27, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 2. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the glutamine (Q) at position 27 binds CD2 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., by at least 5 fold, 10 fold, 20 fold, 30 fold, or 40 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the glutamine (Q) at position 27 binds 2B4 at an increased affinity than aAttorney Docket No: IMID-102WOCD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., by at least 1.5 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells.

[0073] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises an alanine (A) at position 27, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glutamine (Q) at position 27 is to an alanine (A). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 36. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the alanine (A) at position 27 binds CD2 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., by at least 5 fold, 10 fold, 20 fold, 30 fold, or 40 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the alanine (A) at position 27 binds 2B4 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., by at least 1.5 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells.

[0074] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the phenylalanine (F) at position 34, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a tyrosine (Y) at position 34, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the phenylalanine (F) at position 34 is to a tyrosine (Y). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 3. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the phenylalanine (F) at position 34 binds CD2 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., byAttorney Docket No: IMID-102WOat least 2 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells.

[0075] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the lysine (K) at position 86, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises an aspartic acid (D) at position 86, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the lysine (K) at position 86 is to an aspartic acid (D). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 4. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the lysine (K) at position 86 binds CD2 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., by at least 1.5 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells.

[0076] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the serine (S) at position 43, a substitution of the arginine (R) at position 44, a substitution of the lysine (K) at position 45, and a substitution of the serine (S) at position 46, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a valine (V) at position 43, a lysine (K) at position 44, an arginine (R) at position 45, and a leucine (L) at position 46, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the serine (S) at position 43 is to a valine (V), the substitution of the arginine (R) at position 44 is to a lysine (K), the substitution of the lysine (K) at position 45 is to an arginine (R), and the substitution of the serine (S) at position 46 is to a leucine (L). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 6. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the serine (S) at position 43, a substitution of the arginine (R) at position 44, a substitution of the lysine (K) at position 45, and a substitution of the serine (S) at position 46 binds CD2 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., by at least 1.5 fold, measured by flow cytometry to determine the amount of the CD48Attorney Docket No: IMID-102WOIgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the serine (S) at position 43, a substitution of the arginine (R) at position 44, a substitution of the lysine (K) at position 45, and a substitution of the serine (S) at position 46 binds 2B4 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., by at least 1.5 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells.

[0077] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, and a substitution of the threonine (T) at position 78, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises an alanine (A) at position 76, an asparagine (N) at position 77, and an isoleucine (I) at position 78, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the asparagine (N) at position 76 is to an alanine (A), and the substitution of the serine (S) at position 77 is to an asparagine (N), and the substitution of the threonine (T) at position 78 is to an isoleucine (I). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 7. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 7. Without wishing to be bound by theory, it is hypothesized that this NX(S / T) motif, wherein X can be any amino acid except proline (P), is modified by N-linked glycosylation, and elimination of the N-glycosylation site increases the binding of CD48 IgV domain to CD2. Accordingly, other mutations at positions 76-78 that disrupts the NX(S / T) motif are also contemplated. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, and a substitution of the threonine (T) at position 78 binds CD2 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1 , e.g., by at least 5 fold, 10 fold, 20 fold, or 30 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells.

[0078] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glycine (G) at position 88 and a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a methionine (M) at position 88 and a phenylalanine (F) at positionAttorney Docket No: IMID-102WO89, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glycine (G) at position 88 is to a methionine (M) and a substitution of the asparagine (N) at position 89 is to a phenylalanine (F). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 8. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the glycine (G) at position 88 and a substitution of the asparagine (N) at position 89 binds CD2 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., by at least 1.5 fold or 2 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells. In certain embodiments, the CD48 IgV domain variant comprising a substitution of the glycine (G) at position 88 and a substitution of the asparagine (N) at position 89 binds 2B4 at an increased affinity than a CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, e.g., by at least 1.5 fold, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells.

[0079] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, a substitution of the threonine (T) at position 78, a substitution of the glycine (G) at position 88, and a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a glutamic acid (E) at position 27, an alanine (A) at position 76, an asparagine (N) at position 77, an isoleucine (I) at position 78, a methionine (M) at position 88, and a phenylalanine (F) at position 89, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E), the substitution of the asparagine (N) at position 76 is to an alanine (A), the substitution of the serine (S) at position 77 is to an asparagine (N), the substitution of the threonine (T) at position 78 is to an isoleucine (I), the substitution of the glycine (G) at position 88 is to a methionine (M), and the substitution of the asparagine (N) at position 89 is to a phenylalanine (F). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 11. In certainAttorney Docket No: IMID-102WOembodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 11.

[0080] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, a substitution of the phenylalanine (F) at position 34, a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, a substitution of the threonine (T) at position 78, a substitution of the glycine (G) at position 88, and a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a glutamic acid (E) at position 27, a tyrosine (Y) at position 34, an alanine (A) at position 76, an asparagine (N) at position 77, an isoleucine (I) at position 78, a methionine (M) at position 88, and a phenylalanine (F) at position 89, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E), the substitution of the phenylalanine (F) at position 34 is to a tyrosine (Y), the substitution of the asparagine (N) at position 76 is to an alanine (A), the substitution of the serine (S) at position 77 is to an asparagine (N), the substitution of the threonine (T) at position 78 is to an isoleucine (I), the substitution of the glycine (G) at position 88 is to a methionine (M), and the substitution of the asparagine (N) at position 89 is to a phenylalanine (F). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 12. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 12.

[0081] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, a substitution of the threonine (T) at position 78, a substitution of the lysine (K) at position 86, a substitution of the glycine (G) at position 88, and a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a glutamic acid (E) at position 27, an alanine (A) at position 76, an asparagine (N) at position 77, an isoleucine (I) at position 78, an aspartic acid (D) at position 86, a methionine (M) at position 88, and a phenylalanine (F) at position 89, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E), the substitution of the asparagine (N) at position 76 is to an alanine (A), the substitution of the serine (S) at position 77 is to an asparagine (N), the substitution of the threonine (T) atAttorney Docket No: IMID-102WQposition 78 is to an isoleucine (I), the substitution of the lysine (K) at position 86 is to an aspartic acid (D), the substitution of the glycine (G) at position 88 is to a methionine (M), and the substitution of the asparagine (N) at position 89 is to a phenylalanine (F). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 13. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 13.

[0082] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, a substitution of the phenylalanine (F) at position 34, a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, a substitution of the threonine (T) at position 78, a substitution of the lysine (K) at position 86, a substitution of the glycine (G) at position 88, and a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a glutamic acid (E) at position 27, a tyrosine (Y) at position 34, an alanine (A) at position 76, an asparagine (N) at position 77, an isoleucine (I) at position 78, an aspartic acid (D) at position 86, a methionine (M) at position 88, and a phenylalanine (F) at position 89, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E), the substitution of the phenylalanine (F) at position 34 is to a tyrosine (Y), the substitution of the asparagine (N) at position 76 is to an alanine (A), the substitution of the serine (S) at position 77 is to an asparagine (N), the substitution of the threonine (T) at position 78 is to an isoleucine (I), the substitution of the lysine (K) at position 86 is to an aspartic acid (D), the substitution of the glycine (G) at position 88 is to a methionine (M), and a substitution of the asparagine (N) at position 89 is to a phenylalanine (F). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 14. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 14.

[0083] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, a substitution of the phenylalanine (F) at position 34, a substitution of the serine (S) at position 43, a substitution of the arginine (R) at position 44, a substitution of the lysine (K) at position 45, a substitution of the serine (S) at position 46, a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77,Attorney Docket No: IMID-102WOa substitution of the threonine (T) at position 78, a substitution of the lysine (K) at position 86, a substitution of the glycine (G) at position 88, and a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a glutamic acid (E) at position 27, a tyrosine (Y) at position 34, a valine (V) at position 43, a lysine (K) at position 44, an arginine (R) at position 45, and a leucine (L) at position 46, an alanine (A) at position 76, an asparagine (N) at position 77, an isoleucine (I) at position 78, an aspartic acid (D) at position 86, a methionine (M) at position 88, and a phenylalanine (F) at position 89, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E), the substitution of the phenylalanine (F) at position 34 is to a tyrosine (Y), the substitution of the serine (S) at position 43 is to a valine (V), the substitution of the arginine (R) at position 44 is to a lysine (K), the substitution of the lysine (K) at position 45 is to an arginine (R), the substitution of the serine (S) at position 46 is to a leucine (L), the substitution of the asparagine (N) at position 76 is to an alanine (A), the substitution of the serine (S) at position 77 is to an asparagine (N), the substitution of the threonine (T) at position 78 is to an isoleucine (I), the substitution of the lysine (K) at position 86 is to an aspartic acid (D), the substitution of the glycine (G) at position 88 is to a methionine (M), and the substitution of the asparagine (N) at position 89 is to a phenylalanine (F). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%, or 99%) identical to SEQ ID NO: 15. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 15.

[0084] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, a substitution of the serine (S) at position 43, a substitution of the arginine (R) at position 44, a substitution of the lysine (K) at position 45, a substitution of the serine (S) at position 46, a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, a substitution of the threonine (T) at position 78, a substitution of the lysine (K) at position 86, a substitution of the glycine (G) at position 88, and a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a glutamic acid (E) at position 27, a valine (V) at position 43, a lysine (K) at position 44, an arginine (R) at position 45, and a leucine (L) at position 46, an alanine (A) at position 76, an asparagine (N) at position 77, an isoleucine (I) at position 78, an aspartic acid (D) at position 86, aAttorney Docket No: IMID-102WOmethionine (M) at position 88, and a phenylalanine (F) at position 89, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E), the substitution of the serine (S) at position 43 is to a valine (V), the substitution of the arginine (R) at position 44 is to a lysine (K), the substitution of the lysine (K) at position 45 is to an arginine (R), the substitution of the serine (S) at position 46 is to a leucine (L), the substitution of the asparagine (N) at position 76 is to an alanine (A), the substitution of the serine (S) at position 77 is to an asparagine (N), the substitution of the threonine (T) at position 78 is to an isoleucine (I), the substitution of the lysine (K) at position 86 is to an aspartic acid (D), the substitution of the glycine (G) at position 88 is to a methionine (M), and the substitution of the asparagine (N) at position 89 is to a phenylalanine (F). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 37. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 37.

[0085] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, a substitution of the phenylalanine (F) at position 34, a substitution of the serine (S) at position 43, a substitution of the arginine (R) at position 44, a substitution of the lysine (K) at position 45, a substitution of the serine (S) at position 46, a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, a substitution of the threonine (T) at position 78, a substitution of the glycine (G) at position 88, and a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a glutamic acid (E) at position 27, a tyrosine (Y) at position 34, a valine (V) at position 43, a lysine (K) at position 44, an arginine (R) at position 45, and a leucine (L) at position 46, an alanine (A) at position 76, an asparagine (N) at position 77, an isoleucine (I) at position 78, a methionine (M) at position 88, and a phenylalanine (F) at position 89, numbered according to SEQ ID NO: 1. In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E), the substitution of the phenylalanine (F) at position 34 is to a tyrosine (Y), the substitution of the serine (S) at position 43 is to a valine (V), the substitution of the arginine (R) at position 44 is to a lysine (K), the substitution of the lysine (K) at position 45 is to an arginine (R), the substitution of the serine (S) at position 46 is to a leucine (L), the substitution of the asparagine (N) at position 76 is to an alanine (A), the substitution of the serine (S) at position 77 is to an asparagine (N), the substitution of the threonine (T) atAttorney Docket No: IMID-102WOposition 78 is to an isoleucine (I), the substitution of the glycine (G) at position 88 is to a methionine (M), and the substitution of the asparagine (N) at position 89 is to a phenylalanine (F). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 38. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 38.

[0086] In certain embodiments, the CD48 IgV domain variant comprises a substitution of the glutamine (Q) at position 27, a substitution of the serine (S) at position 43, a substitution of the arginine (R) at position 44, a substitution of the lysine (K) at position 45, a substitution of the serine (S) at position 46, a substitution of the asparagine (N) at position 76, a substitution of the serine (S) at position 77, a substitution of the threonine (T) at position 78, a substitution of the glycine (G) at position 88, and a substitution of the asparagine (N) at position 89, relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant comprises a glutamic acid (E) at position 27, a valine (V) at position 43, a lysine (K) at position 44, an arginine (R) at position 45, and a leucine (L) at position 46, an alanine (A) at position 76, an asparagine (N) at position 77, an isoleucine (I) at position 78, a methionine (M) at position 88, and a phenylalanine (F) at position 89, numbered according to SEQ ID NO: 1 In certain embodiments, the substitution of the glutamine (Q) at position 27 is to a glutamic acid (E), the substitution of the serine (S) at position 43 is to a valine (V), the substitution of the arginine (R) at position 44 is to a lysine (K), the substitution of the lysine (K) at position 45 is to an arginine (R), the substitution of the serine (S) at position 46 is to a leucine (L), the substitution of the asparagine (N) at position 76 is to an alanine (A), the substitution of the serine (S) at position 77 is to an asparagine (N), the substitution of the threonine (T) at position 78 is to an isoleucine (I), the substitution of the glycine (G) at position 88 is to a methionine (M), and the substitution of the asparagine (N) at position 89 is to a phenylalanine (F). In certain embodiments, the CD48 IgV domain variant comprises an amino acid sequence at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 5. In certain embodiments, the CD48 IgV domain variant comprises the amino acid sequence of SEQ ID NO: 5.

[0087] Also contemplated at one or more of these positions are one or more alternative amino acid residues conservative to the substitutions identified above, e.g., having a positive score between the original substitution (e.g., glutamic acid (E) at position 27) and anAttorney Docket No: IMID-102WOalternative amino acid residue under a scoring matrix suitable for CD48 IgV sequence, such as BLOSUM-62. Table 2 identifies the amino acid residues that are conservative to each other, according to the scoring matrix of BLOSUM-62.

[0088] In certain embodiments, where the present disclosure identifies a substitution of the glutamine (Q) at position 27 to a glutamic acid (E), also contemplated are substitution of the glutamine (Q) at position 27 to an aspartic acid (D) or a lysine (K). In certain embodiments, where the present disclosure identifies a substitution of the phenylalanine (F) at position 34 to a tyrosine (Y), also contemplated are substitution of the phenylalanine (F) at position 34 to a tryptophan (W) or a histidine (H). In certain embodiments, where the present disclosure identifies a substitution of the serine (S) at position 43 to a valine (V), also contemplated are substitution of the serine (S) at position 43 to a methionine (M), an isoleucine (I), or a leucine (L). In certain embodiments, where the present disclosure identifies a substitution of the arginine (R) at position 44 to a lysine (K), also contemplated is substitution of the arginine (R) at position 44 to a glutamic acid (E) or a glutamine (Q). In certain embodiments, where the present disclosure identifies a substitution of the lysine (K) at position 45 to an arginine (R), also contemplated is substitution of the lysine (K) at position 45 to a glutamine (Q). In certain embodiments, where the present disclosure identifies a substitution of the serine (S) at position 46 to a leucine (L), also contemplated are substitution of the serine (S) at position 46 to a methionine (M), an isoleucine (I), or a valine (V). In certain embodiments, where the present disclosure identifies a substitution of the asparagine (N) at position 76 to an alanine (A), also contemplated is substitution of the asparagine (N) at position 76 to a serine (S). In certain embodiments, where the present disclosure identifies a substitution of the serine (S) at position 77 to an asparagine (N), also contemplated are substitution of the serine (S) at position 77 to a histidine (H) or an aspartic acid (D). In certain embodiments, where the present disclosure identifies a substitution of the threonine (T) at position 78 to an isoleucine (I), also contemplated are substitution of the threonine (T) at position 78 to a methionine (M), a leucine (L), or a valine (V). In certain embodiments, where the present disclosure identifies a substitution of the lysine (K) at position 86 to an aspartate acid (D), also contemplated are substitution of the lysine (K) position 86 to an asparagine (N) or a glutamate acid (E). In certain embodiments, where the present disclosure identifies a substitution of the glycine (G) at position 88 to a methionine (M), also contemplated are substitution of the glycine (G) at position 88 to an isoleucine (I), a leucine (L), or a valine (V). In certain embodiments, where the present disclosure identifies aAttorney Docket No: IMID-102WOsubstitution of the asparagine (N) at position 89 to a phenylalanine (F), also contemplated are substitution of the asparagine (N) at position 89 to a tryptophan (W) or a tyrosine (Y). The alternative substitutions can be combined with the original substitutions and / or one another to generate more CD48 IgV domain variants.Table 2: Conservative Amino Acid Substitutions under BLOSUM-62 Matrix

[0089] It has been observed that the interaction surfaces of CD48:2B4 and CD58:CD2 are highly hydrophilic. It is a surprising discovery that hydrophobic substitutions in human CD48, such as S43V, R44K, K45R, S46L, N76A, S77N, T78I, G88M, and N89F, can improve its binding affinities to CD2 and / or 2B4. It is therefore contemplated that the amino acids at one or more positions selected from 43-46, 76-78, and 88-89, relative to SEQ ID NO: 1, can be mutated to one or more hydrophobic amino acids. In certain embodiments, where the present disclosure identifies a substitution of the serine (S) at position 43 to a valine (V), also contemplated are substitution of the serine (S) at position 43 to a methionine (M), anAttorney Docket No: IMID-102WOisoleucine (I), or a leucine (L). In certain embodiments, where the present disclosure identifies a substitution of the serine (S) at position 46 to a leucine (L), also contemplated are substitution of the serine (S) at position 46 to a methionine (M), an isoleucine (I), or a valine (V). In certain embodiments, where the present disclosure identifies a substitution of the threonine (T) at position 78 to an isoleucine (I), also contemplated are substitution of the threonine (T) at position 78 to a methionine (M), a leucine (L), or a valine (V). In certain embodiments, where the present disclosure identifies a substitution of the glycine (G) at position 88 to a methionine (M), also contemplated are substitution of the glycine (G) at position 88 to an isoleucine (I), a leucine (L), or a valine (V). In certain embodiments, where the present disclosure identifies a substitution of the asparagine (N) at position 89 to a phenylalanine (F), also contemplated is substitution of the asparagine (N) at position 89 to a tryptophan (W). The alternative substitutions can be combined with the original substitutions and / or one another to generate more CD48 IgV domain variants.

[0090] In some embodiments, the CD48 IgV domain variant comprises one or more additional mutations (e.g., substitutions) relative to SEQ ID NO: 1. The substituted amino acid residue(s) can be, but are not necessarily, conservative substitutions, under BLOSUM-62 scoring matrix. Exemplary CD48 IgV variants can include conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0091] In certain embodiments, the CD48 IgV domain variant, when present as a homodimer (e.g., fused to an Fc dimer), binds CD2 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 5 pM, 2 pM, 1 pM, 500 nM. 200 nM, 100 nM, 90 nM, 80 nM, or 70 nM, as measured by surface plasmon resonance (SPR). In certain embodiments, the CD48 IgV domain variant, when present as a homodimer (e.g., fused to an Fc dimer), binds 2B4 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM, as measured by SPR. In certain embodiments, the CD48 IgV domain variant, when present as a homodimer (e.g., fused to an Fc dimer), binds CD2 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 2 pM and binds 2B4 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 5 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM, as measured by SPR. In certain embodiments, the CD48 IgV domain variant, when present as a homodimer (e.g., fused to an Fc dimer), binds CD2 or anAttorney Docket No: IMID-102WOextracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 1 pM and binds 2B4 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 5 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM, as measured by SPR. In certain embodiments, the CD48 IgV domain variant, when present as a homodimer (e.g., fused to an Fc dimer), binds CD2 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 500 nM and binds 2B4 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 5 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM, as measured by SPR. In certain embodiments, the CD48 IgV domain variant, when present as a homodimer (e.g., fused to an Fc dimer), binds CD2 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 200 nM and binds 2B4 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 5 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM, as measured by SPR. In certain embodiments, the CD48 IgV domain variant, when present as a homodimer (e.g., fused to an Fc dimer), binds CD2 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 100 nM and binds 2B4 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 5 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM, as measured by SPR. In certain embodiments, the CD48 IgV domain variant, when present as a homodimer (e.g., fused to an Fc dimer), binds CD2 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 70 nM and binds 2B4 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 5 nM, 2 nM, 1 nM, 0.5 nM, or 0.25 nM, as measured by SPR. In certain embodiments, the CD48 IgV domain variant, when present as a homodimer (e.g., fused to an Fc dimer), binds CD2 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 70 nM and binds 2B4 or an extracellular fragment thereof at a dissociation constant (KD) value smaller than or equal to (affinity greater than or equal to) 1 nM, 0.5 nM, or 0.25 nM, as measured by SPR.

[0092] In certain embodiments, the CD48 IgV domain variant binds at least one of CD2 and 2B4, or an extracellular fragment thereof, at an affinity at least 1.1, 1.2, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher than a wild-type human CD48 IgV domainAttorney Docket No: IMID-102WOhaving the amino acid sequence of SEQ ID NO: 1, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to cells expressing the respective antigens (CD2 and / or 2B4). In certain embodiments, the CD48 IgV domain variant binds at least one of CD2 and 2B4, or an extracellular fragment thereof, at about 1.1 fold to about 200 fold, about 2 fold to about 200 fold, about 5 fold to about 200 fold, about 10 fold to about 200 fold, about 20 fold to about 200 fold, about 30 fold to about 200 fold, about 40 fold to about 200 fold, about 50 fold to about 200 fold, about 75 fold to about 200 fold, about 90 fold to about 200 fold, at about 1.1 fold to about 150 fold, about 2 fold to about 150 fold, about 5 fold to about 150 fold, about 10 fold to about 150 fold, about 20 fold to about 150 fold, about 30 fold to about 150 fold, about 40 fold to about 150 fold, about 50 fold to about 150 fold, about 75 fold to about 150 fold, about 90 fold to about 150 fold, about 1.1 fold to about 110 fold, about 2 fold to about 110 fold, about 5 fold to about 110 fold, about 10 fold to about 110 fold, about 20 fold to about 110 fold, about 30 fold to about 110 fold, about 40 fold to about 110 fold, about 50 fold to about 110 fold, about 75 fold to about 110 fold, about 90 fold to about 110 fold, about 1.1 fold to about 80 fold, about 2 fold to about 80 fold, about 5 fold to about 80 fold, about 10 fold to about 80 fold, about 20 fold to about 80 fold, about 40 fold to about 80 fold, about 60 fold to about 80 fold, about 1.1 fold to about 40 fold, about 2 fold to about 40 fold, about 10 fold to about 40 fold, or about 20 fold to about 40 fold higher affinity than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to cells expressing the respective antigens (CD2 and / or 2B4).

[0093] In certain embodiments, the CD48 IgV domain variant binds at least one of CD2 and 2B4, or an extracellular fragment thereof, at an affinity at least 1.1, 1.2, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher than a corresponding CD48 IgV domain not having the one or more mutations, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to cells expressing the respective antigens (CD2 and / or 2B4). In certain embodiments, the CD48 IgV domain variant binds at least one of CD2 and 2B4, or an extracellular fragment thereof, at about 1.1 fold to about 200 fold, about 2 fold to about 200 fold, about 5 fold to about 200 fold, about 10 fold to about 200 fold, about 20 fold to about 200 fold, about 30 fold to about 200 fold, about 40 fold to about 200 fold, about 50 fold to about 200 fold, about 75 fold to about 200 fold, about 90 fold to about 200 fold, at about 1.1 fold to about 150 fold, about 2 fold to about 150Attorney Docket No: IMID-102WOfold, about 5 fold to about 150 fold, about 10 fold to about 150 fold, about 20 fold to about 150 fold, about 30 fold to about 150 fold, about 40 fold to about 150 fold, about 50 fold to about 150 fold, about 75 fold to about 150 fold, about 90 fold to about 150 fold, about 1.1 fold to about 110 fold, about 2 fold to about 110 fold, about 5 fold to about 110 fold, about 10 fold to about 110 fold, about 20 fold to about 110 fold, about 30 fold to about 110 fold, about 40 fold to about 110 fold, about 50 fold to about 110 fold, about 75 fold to about 110 fold, about 90 fold to about 110 fold, about 1.1 fold to about 80 fold, about 2 fold to about 80 fold, about 5 fold to about 80 fold, about 10 fold to about 80 fold, about 20 fold to about 80 fold, about 40 fold to about 80 fold, about 60 fold to about 80 fold, about 1.1 fold to about 40 fold, about 2 fold to about 40 fold, about 10 fold to about 40 fold, or about 20 fold to about 40 fold higher affinity than a corresponding CD48 IgV domain not having the one or more mutations, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to cells expressing the respective antigens (CD2 and / or 2B4).

[0094] In certain embodiments, the CD48 IgV domain variant binds CD2, or an extracellular fragment thereof, at an affinity at least 1.1, 1.2, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at about 1.1 fold to about 200 fold, about 2 fold to about 200 fold, about 5 fold to about 200 fold, about 10 fold to about 200 fold, about 20 fold to about 200 fold, about 30 fold to about 200 fold, about 40 fold to about 200 fold, about 50 fold to about 200 fold, about 75 fold to about 200 fold, about 90 fold to about 200 fold, at about 1.1 fold to about 150 fold, about 2 fold to about 150 fold, about 5 fold to about 150 fold, about 10 fold to about 150 fold, about 20 fold to about 150 fold, about 30 fold to about 150 fold, about 40 fold to about 150 fold, about 50 fold to about 150 fold, about 75 fold to about 150 fold, about 90 fold to about 150 fold, about 1.1 fold to about 110 fold, about 2 fold to about 110 fold, about 5 fold to about 110 fold, about 10 fold to about 110 fold, about 20 fold to about 110 fold, about 30 fold to about 110 fold, about 40 fold to about 110 fold, about 50 fold to about 110 fold, about 75 fold to about 110 fold, about 90 fold to about 110 fold, about 1.1 fold to about 80 fold, about 2 fold to about 80 fold, about 5 fold to about 80 fold, about 10 fold to about 80 fold, about 20 fold to about 80 fold, about 40 fold to about 80 fold, about 60 fold to about 80 fold, about 1.1 fold to about 40 fold, about 2 fold to about 40 fold, about 10 fold to about 40Attorney Docket No: IMID-102WOfold, or about 20 fold to about 40 fold higher affinity than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells.

[0095] In certain embodiments, the CD48 IgV domain variant binds CD2, or an extracellular fragment thereof, at an affinity at least 1.1, 1.2, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher than a corresponding CD48 IgV domain not having the one or more mutations, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at about 1.1 fold to about 200 fold, about 2 fold to about 200 fold, about 5 fold to about 200 fold, about 10 fold to about 200 fold, about 20 fold to about 200 fold, about 30 fold to about 200 fold, about 40 fold to about 200 fold, about 50 fold to about 200 fold, about 75 fold to about 200 fold, about 90 fold to about 200 fold, at about 1.1 fold to about 150 fold, about 2 fold to about 150 fold, about 5 fold to about 150 fold, about 10 fold to about 150 fold, about 20 fold to about 150 fold, about 30 fold to about 150 fold, about 40 fold to about 150 fold, about 50 fold to about 150 fold, about 75 fold to about 150 fold, about 90 fold to about 150 fold, about 1.1 fold to about 110 fold, about 2 fold to about 110 fold, about 5 fold to about 110 fold, about 10 fold to about 110 fold, about 20 fold to about 110 fold, about 30 fold to about 110 fold, about 40 fold to about 110 fold, about 50 fold to about 110 fold, about 75 fold to about 110 fold, about 90 fold to about 110 fold, about 1.1 fold to about 80 fold, about 2 fold to about 80 fold, about 5 fold to about 80 fold, about 10 fold to about 80 fold, about 20 fold to about 80 fold, about 40 fold to about 80 fold, about 60 fold to about 80 fold, about 1.1 fold to about 40 fold, about 2 fold to about 40 fold, about 10 fold to about 40 fold, or about 20 fold to about 40 fold higher affinity than a corresponding CD48 IgV domain not having the one or more mutations, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells.

[0096] In certain embodiments, the CD48 IgV domain variant binds 2B4, or an extracellular fragment thereof, at an affinity at least 1.1, 1.2, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5 fold higher than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells. In certainAttorney Docket No: IMID-102WOembodiments, the CD48 IgV domain variant binds 2B4, or an extracellular fragment thereof, at an affinity at least 2, 3, or 4 fold higher than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells. In certain embodiments, the CD48 IgV domain variant binds 2B4 or an extracellular fragment thereof at about 1.1 fold to about 5 fold, about 1.5 fold to about 5 fold, about 2 fold to about 5 fold, about 2.5 fold to about 5 fold, about 3 fold to about 5 fold, about 1.1 fold to about 4 fold, about 1.5 fold to about 4 fold, about 2 fold to about 4 fold, about 2.5 fold to about 4 fold, about 3 fold to about 4 fold, about 1.1 fold to about 3.5 fold, about 1.5 fold to about 3.5 fold, about 2 fold to about 3.5 fold, about 2.5 fold to about 3.5 fold, or about 3 fold to about 3.5 fold higher affinity than a wild-type human CD48 IgV protein represented by SEQ ID NO: 1, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells.

[0097] In certain embodiments, the CD48 IgV domain variant binds 2B4, or an extracellular fragment thereof, at an affinity at least 1.1, 1.2, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5 fold higher than a corresponding CD48 IgV domain not having the one or more mutations, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells. In certain embodiments, the CD48 IgV domain variant binds 2B4, or an extracellular fragment thereof, at an affinity at least 2, 3, or 4 fold higher than a corresponding CD48 IgV domain not having the one or more mutations, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells. In certain embodiments, the CD48 IgV domain variant binds 2B4 or an extracellular fragment thereof at about 1.1 fold to about 5 fold, about 1.5 fold to about 5 fold, about 2 fold to about 5 fold, about 2.5 fold to about 5 fold, about 3 fold to about 5 fold, about 1.1 fold to about 4 fold, about 1.5 fold to about 4 fold, about 2 fold to about 4 fold, about 2.5 fold to about 4 fold, about 3 fold to about 4 fold, about 1.1 fold to about 3.5 fold, about 1.5 fold to about 3.5 fold, about 2 fold to about 3.5 fold, about 2.5 fold to about 3.5 fold, or about 3 fold to about 3.5 fold higher affinity than a corresponding CD48 IgV domain not having the one or more mutations, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to 2B4-expressing cells.Attorney Docket No: IMID-102WQ

[0098] In certain embodiments, the CD48 IgV domain variant binds both CD2 and 2B4, or extracellular fragments thereof, at increased affinities than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at an affinity at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher, and binds 2B4 or an extracellular fragment thereof at an affinity at least 2, 3, or 4 fold higher, than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells and 2B4-expressing cells, respectively. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at an affinity at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher, and binds 2B4 or an extracellular fragment thereof at an affinity at least 2 fold higher, than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at an affinity at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher, and binds 2B4 or an extracellular fragment thereof at an affinity at least 3 fold higher, than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at an affinity at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher, and binds 2B4 or an extracellular fragment thereof at an affinity at least 4 fold higher, than a wild-type human CD48 IgV domain having the amino acid sequence of SEQ ID NO: 1.

[0099] In certain embodiments, the CD48 IgV domain variant binds both CD2 and 2B4, or extracellular fragments thereof, at increased affinities than a corresponding CD48 IgV domain not having the one or more mutations, measured by flow cytometry to determine the amount of the CD48 IgV domain variant, in an Fc fused dimeric form, bound to CD2-expressing cells and 2B4-expressing cells, respectively. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at an affinity at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher, and binds 2B4 or an extracellular fragment thereof at an affinity at least 2, 3, or 4 fold higher, than a corresponding CD48 IgV domain not having the one or more mutations. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at an affinity at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher, and binds 2B4 or an extracellular fragment thereof at an affinity at least 2 fold higher, than a corresponding CD48 IgV domain not having the oneAttorney Docket No: IMID-102WOor more mutations. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at an affinity at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher, and binds 2B4 or an extracellular fragment thereof at an affinity at least 3 fold higher, than a corresponding CD48 IgV domain not having the one or more mutations. In certain embodiments, the CD48 IgV domain variant binds CD2 or an extracellular fragment thereof at an affinity at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold higher, and binds 2B4 or an extracellular fragment thereof at an affinity at least 4 fold higher, than a corresponding CD48 IgV domain not having the one or more mutations.B. CD48-Fc Fusion Proteins

[0100] In another aspect, the present disclosure provides a CD48 fusion protein (e.g., a soluble CD48 fusion protein). In some embodiments, the fusion protein comprises a CD48 fragment and an IgG Fc region. The IgG Fc region may facilitate dimerization of the CD48 fragment and extend its serum half-life. Other CD48 fusion proteins are also contemplated by the present disclosure. For example, Fc regions of other immunoglobulins, such as IgM, IgA, IgD, and IgE, may also be fused with a CD48 fragment to facilitate dimerization or multimerization. Serum albumin proteins and antigen-binding domains that bind serum albumins may also be fused with a CD48 fragment to extend its serum half-life.CD48 Fragments

[0101] In another aspect, the present disclosure provides a CD48 protein or fragment thereof comprising a CD48 IgV domain variant. The CD48 IgV domain variant can comprise any of the amino acid mutations disclosed herein. In some embodiments, the CD48 fragment comprises the IgV domain variant and an Ig constant-like (IgC2) domain.

[0102] In certain embodiments, the IgC2 domain is a wild-type human IgC2 domain comprising the amino acid sequence of PKPVIKIEKIEDMDDNCYLKLSCVIPGESVNYTWYGDKRPFPKELQNSVLETTLMPH NYSRCYTCQVSNSVSSKNGTVCLS (SEQ ID NO: 17). In certain embodiments, the IgC2 domain comprises an amino acid sequence at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17. In certain embodiments, the CD48 IgC2 domain comprises one or more additional conservative substitutions relative to SEQ ID NO: 17.Attorney Docket No: IMID-102WO

[0103] In certain embodiments, the IgV domain variant is linked to an N-terminus of the IgC2 domain, either directly (e.g., by a peptide bond) or via a linker. The linker can comprise or consist of the amino acid sequence of LDPV (SEQ ID NO: 18). Alternatively, the linker can be a linker described in the subsection titled “Linkers” below, e.g., comprising an amino acid sequence in Table 4. In some embodiments, the CD48 protein or fragment thereof is anchored to a plasma membrane (e.g., by a GPI linkage). In some embodiments, the CD48 fragment thereof is soluble.Fc regions

[0104] A CD48-Fc fusion polypeptide of the present disclosure can comprise a CD48 fragment (e.g., one comprising a CD48 IgV domain variant disclosed herein) linked to an IgG Fc region, either directly (e.g., by a peptide bond) or via a linker. In certain embodiments, the CD48 fragment comprises a CD48 IgV domain variant and a CD48 IgC2 domain.

[0105] In certain embodiments, the IgG Fc region is an IgGl, IgG2, IgG3, or IgG4 Fc region, e.g., a wild-type human IgGl, IgG2, IgG3, or IgG4 Fc region or a variant thereof. An IgG Fc region generally includes a lower hinge, CH2 and CH3 domains of an IgG. Two IgG Fc regions can form an Fc dimer. Boundaries between IgG hinge regions, CH2, and CH3 domains are well known in the art, and can be found, e.g., in the PROSITE database (prosite.expasy.org). In certain embodiments, the IgG Fc region is a human IgGl Fc region. In certain embodiments, the IgG Fc region is a human IgG4 Fc region (e.g., a human IgG4 Fc region with an S228P mutation).

[0106] Certain IgG Fc regions have one or more effector functions in its wild-type form. For example, wild-type human IgGl Fc region can mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). ADCC is generally mediated by Fc receptors such as FcyRIIIA (CD 16), and CDC is generally mediated by one or more complement components such as Cl q, Cl r, and / or Cis. The binding of Fc region to CD 16 is mediated, at least in part, by the hinge and the CH2 domain. For example, within human IgGl, the interaction with CD 16 involves amino acid residues Asp 265 - Glu 269, Asn 297 - Thr 299, Ala 327 - He 332, Leu 234 - Ser 239, and carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see, Sondermann et al., (2000) NATURE, 406 (6793):267-273). The binding of Fc region to Clq is mediated, at least in part, by the CH2 domain. For example, within human IgGl, the interaction with Clq involves amino acid residues Glu 318,Attorney Docket No: IMID-102WOLys 320, Lys 322, and Pro 331 (see Ward and Ghetie (1995) THERAPEUTIC IMMUNOLOGY 2:77-94).

[0107] One or more mutations can be introduced to the Fc region to increase, decrease, or abrogate the effector functions, e.g., by enhancing or reducing the interaction of the Fc region with the one or more Fc receptors (e.g., GDI 6) and / or complement components (e.g., Clq). Exemplary Fc mutations are disclosed in International Application Publication No.WO1999 / 051642. In some embodiments, the CD48-Fc fusion protein comprises a human IgGl Fc sequence comprising one or more mutations to reduce effector functions. For example, in certain embodiments, the one or more mutations comprise N297A, N297G, or N297Q mutation. In certain embodiments, the one or more mutations comprises or further comprises L234A and L235A (LALA) mutations, L234A, L235A, and P329A (LALAPA) mutations, L234A, L235A, and P329G (LALAPG) mutations, L234A, L235E, G237A, A330S, and P331S (LALEGAASPS) mutations, or L234F, L235Q, and K322Q mutations.

[0108] Exemplary Fc region amino acid sequences are provided in Table 3. In certain embodiments, the Fc region comprises an amino acid sequence of SEQ ID NO: 19, 20, 21, 22, or 23. In certain embodiments, the Fc region has no substantial ADCC effector function and comprises an amino acid sequence of SEQ ID NO: 20, 21, 22, or 23.Table 3: Exemplary Fc VariantsAttorney Docket No: IMID-102WO*The amino acid sequences can further comprise a lysine (K) at the C-terminus.

[0109] Fc regions are known to extend serum half-life of proteins conjugated or fused thereto. In certain embodiments, the CD48-Fc fusion protein has a longer serum half-life than the corresponding CD48 fragment alone not fused to an Fc region. In certain embodiments, the CD48-Fc fusion protein has a serum half-life that is at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, or at least 20 fold longer than the corresponding CD48 fragment alone not fused to an Fc region.

[0110] The IgG Fc region can comprise one or more mutations that increase its binding to the Neonatal Fc Receptor (FcRn), thereby to extend the serum half-life of the CD48-Fc fusion protein. Such mutations are known in the art and reviewed by Ramdani et al. , INT. J. MOL. Set. (2022) 23(17): 9604. In certain embodiments, the one or more mutations comprise M252Y, S254T, and T256E (YTE); M428L and N434S (Xtend™); H433K and N434FAttorney Docket No: IMID-102WO(NHance®); M252Y and T256D; T250Q and M428L; T307A, E380A, and N434A; T256D and T307Q, or T256D and T307W.

[0111] In certain embodiments, the CD48-Fc fusion protein has a longer serum half-life than the corresponding CD48 fragment fused to a corresponding wild-type Fc region. In certain embodiments, the CD48-Fc fusion protein has a serum half-life that is at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, or at least 20 fold longer than the corresponding CD48 fragment fused to a corresponding wild-type Fc region.

[0112] In certain embodiments, the CD48-Fc fusion protein comprise an Fc region (e.g., a human IgGl Fc region) comprising one or more mutations that reduce effector functions and one or more mutations that increase binding to FcRn. In certain embodiments, the one or more mutations that reduce effector functions comprise L234A and L235A, and the one or more mutations that increase binding to FcRn comprise M252Y, S254T, and T256E. In certain embodiments, the one or more mutations that reduce effector functions comprise L234F, L235Q, and K322Q, and the one or more mutations that increase binding to FcRn comprise M252Y, S254T, and T256E.

[0113] IgG Fc regions generally form dimers. Accordingly, the present disclosure also provides a CD48-Fc fusion protein comprising a first CD48-Fc fusion polypeptide and a second CD48-Fc fusion polypeptide. The two fusion polypeptides can be covalently or non-covalently associated with one another. In one embodiment, the two fusion polypeptides can be covalently linked together by, for example, one or more disulfide bonds. In certain embodiments, the first and second CD48-Fc fusion polypeptides comprise the same mutation(s) that reduce ADCC and / or CDC effector functions. In certain embodiments, the first and second CD48-Fc fusion polypeptides comprise the same mutation(s) that extend serum half-life. In certain embodiments, the CD48-Fc fusion protein is a homodimer, wherein the first and second CD48-Fc fusion polypeptides comprise the same amino acid sequence. In certain embodiments, in the homodimer, each polypeptide chain comprises, from N-terminus to C-terminus, a CD48 IgV domain variant, a CD48 IgC2 domain, and an IgG Fc region. The structure of this homodimer is illustrated in FIG. 1.

[0114] Heterodimeric CD48-Fc fusion proteins are also contemplated. Such protein can have the advantage of comprising asymmetric structures, such as a single copy of a moiety conjugated to only the first CD48-Fc fusion polypeptide or only the second CD48-Fc fusionAttorney Docket No: IMID-102WOpolypeptide. Preferential assembly of Fc heterodimers can be accomplished by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region as shown in U.S. 8,679,785, U.S. 7,695,936, U.S. 9,493,578, U.S. 9,562,109, U.S. 10,047,167, U.S. 8,592,562, U.S. 9,248,182, U.S. 9,951,145, and U.S. 2016 / 0046727. For example, the knob-in-holes mutations comprise T366W mutation in one Fc chain and T366S, L368A, and Y407V mutations in the other Fc chain. In certain embodiments, the CD48-Fc fusion protein comprises Fc mutations that promote heterodimer formation.

[0115] A skilled person in the art would appreciate that during production and / or storage of proteins, N-terminal glutamate (E) or glutamine (Q) can be cyclized to form a lactam (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage).Accordingly, in some embodiments where the N-terminal residue of an amino acid sequence of a polypeptide is E or Q, a corresponding amino acid sequence with the E or Q replaced with pyroglutamate is also contemplated herein.

[0116] A skilled person in the art would also appreciate that during protein production and / or storage, the C-terminal lysine (K) of a protein can be removed (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Such removal of K is often observed with proteins that comprise a Fc domain at its C-tenninus. Accordingly, in some embodiments where the C-terminal residue of an amino acid sequence of a polypeptide (e.g., a Fc domain sequence) is K, a corresponding amino acid sequence with the K removed is also contemplated herein.

[0117] It is contemplated that the Fc regions may improve the yield of the protein during production, allow better purification methods in the manufacturing process (e.g., protein A chromatography), enhance protein stability during storage, and / or increase the serum half-life of the protein after administration to a subject. Furthermore, the dimerization may increase the avidity of the protein to a CD48 receptor, such as 2B4 or CD2.Linkers

[0118] In certain embodiments, the CD48 fragment disclosed herein is fused to an IgG Fc region via a linker (e.g., peptide linker). In certain embodiments, a C-terminus of the CD48 fragment is linked to an N-terminus of the IgG Fc region via the linker. In certain embodiments, a CD48 IgV domain variant disclosed herein is linked a CD48 IgC2 domain via a linker (e.g., peptide linker). In certain embodiments, a C-terminus of the CD48 IgV domain variant is linked an N-terminus of the CD48 IgC2 domain via the linker.Attorney Docket No: IMID-102WO

[0119] The peptide linker can have an optimized length and / or amino acid composition to be flexible or rigid. In certain embodiments, the peptide linker includes 4-50 amino acid residues. In certain embodiments, the peptide linker includes 4-20 amino acid residues. In certain embodiments, the peptide linker is short, e.g., consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, the peptide linker consists of about 12 or fewer amino acid residues. In certain embodiments, the peptide linker is long, e.g., consists of 15-50, 15-25, 15-20, 20-50, 20-25, or 25-50 amino acid residues. In some embodiments, the peptide linker consists of 3 to 15, for example, 8, 9 or 10 contiguous amino acid residues. Regarding the amino acid composition of the peptide linker, peptides are selected with properties that confer flexibility, do not interfere with the binding of the domains or regions linked to their respective receptors, and resist cleavage from proteases.For example, glycine and serine residues generally provide protease resistance. The peptide linker, e.g., suitable for linking the CD48 IgV fragment to the Fc region, can comprise a (GS)n, (GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, or (GGGGS)n sequence, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. or 20 (SEQ ID NOs: 43-48, respectively, in order of appearance). Exemplary peptide linker sequences are provided in Table 4. In certain embodiments, a C-terminus of the CD48 fragment is fused to an N-terminus of an IgG Fc region (e.g., any one of the human IgG Fc regions described in the subsection titled “Fc regions” above) by a peptide linker listed in Table 4.Table 4: Peptide LinkersAttorney Docket No: IMID-102WOExemplary CD48-Fc Fusion Proteins

[0120] The present disclosure provides two exemplary CD48-Fc fusion proteins, namely, CD48-C5-Fc and CD48-mI-Fc, each containing a variant of CD48 IgV domain.

[0121] CD48-C5-Fc is a homodimer comprising two polypeptides, each having the amino acid sequence of SEQ ID NO: 39. The sequence of SEQ ID NO: 39 comprises a mature human CD48 polypeptide (without the propeptide or the GPI anchor) fused with human IgGl hinge, CH2 and CH3 domains via a peptide linker having the amino acid sequence of SEQ ID NO: 9. The mature human CD48 polypeptide comprises an IgV domain comprising Q27E, F34Y, S43V, R44K, K45R, S46L, N76A, S77N, T78I, K86D, G88M, and N89F substitutions, as present in the C5 variant, and an IgC2 domain of human CD48. The hinge comprises L234A and L235A substitutions to reduce effector functions. The CH2 domain comprises M252Y, S254T, and T256E substitutions to increase binding to FcRn, thereby to extend serum half-life of the fusion protein. The substitutions in the hinge and CH2 domain are bolded and underlined in Table 5 below.

[0122] CD48-mI-Fc is a homodimer comprising two polypeptides, each having the amino acid sequence of SEQ ID NO: 40. The sequence of SEQ ID NO: 40 comprises a mature human CD48 polypeptide (without the propeptide or the GPI anchor) fused with human IgGl hinge, CH2 and CH3 domains via a peptide linker having the amino acid sequence of SEQ ID NO: 9. The mature human CD48 polypeptide comprises an IgV domain comprising Q27E, F34Y, S43V, R44K, K45R, S46L, N76A, S77N, T78I, G88M, and N89F substitutions, as present in the ml variant, and an IgC2 domain of human CD48. The hinge comprises L234A and L235A substitutions to reduce effector functions. The CH2 domain comprises M252Y, S254T, and T256E substitutions to increase binding to FcRn, thereby to extend serum halflife of the fusion protein. The substitutions in the hinge and CH2 domain are bolded and underlined in Table 5 below.Attorney Docket No: IMID-102WOTable 5: Exemplary CD48-Fc fusion protein sequences

[0123] In certain embodiments, a CD48-Fc fusion protein disclosed herein comprises a polypeptide comprising an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 39, optionally further comprising a C-terminal lysine residue. In certain embodiments, a CD48-Fc fusion protein disclosed herein comprises two polypeptide each comprising an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 39, optionally further comprising a C-terminal lysine residue. In certain embodiments, the two polypeptide have identical amino acid sequences.

[0124] In certain embodiments, a CD48-Fc fusion protein disclosed herein comprises a polypeptide comprising an amino acid sequence at least 80%, at least 81%, at least 82%, atAttorney Docket No: IMID-102WOleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 40, optionally further comprising a C-terminal lysine residue. In certain embodiments, a CD48-Fc fusion protein disclosed herein comprises two polypeptide each comprising an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 40, optionally further comprising a C-terminal lysine residue. In certain embodiments, the two polypeptide have identical amino acid sequences.III. Methods of Protein Production

[0125] The proteins of the present disclosure can be made using recombinant DNA technology well known to a skilled person in the art. For example, a nucleic acid sequence encoding a CD48 IgV domain variant or a CD48 fragment disclosed herein can be used cloned into an expression vector, stably transfected into host cells to produce the CD48 IgV domain variant or the CD48 fragment. For producing a homodimeric CD48-Fc fusion protein, a nucleic acid sequence encoding a CD48-Fc fusion polypeptide can be cloned into an expression vector, stably transfected into host cells, wherein two CD48-Fc fusion polypeptide can pair to produce the homodimeric CD48-Fc fusion protein. A signal peptide can be fused to an N-terminus of the CD48 IgV domain variant, the CD48 fragment, or the CD48-Fc fusion polypeptide, to allow secretion of the mature protein into the culture medium of the host cells. To produce such mature protein recombinantly, the nucleic acid sequence can encode a corresponding immature polypeptide sequence that includes the signal peptide.

[0126] Nucleic acids encoding desired recombinant proteins disclosed herein can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. Where the recombinant protein comprises an IgG Fc region, it is understood that Fc affinity chromatography may be used for protein purification. Accordingly, the host cells above, which do not otherwise produce IgG proteins, are particularly suitable for recombinantly producing Fc-fusionAttorney Docket No: IMID-102WOproteins. Transformed host cells can be grown under conditions that permit the host cells to express the gene that encodes the CD48 IgV domain variant, CD48 fragment, or CD48-Fc fusion polypeptide.

[0127] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed protein may be secreted. Alternatively, the expressed protein may accumulate in refractile or inclusion bodies, which can be harvested after disruption of the cells by French press or sonication. The refractile or inclusion bodies then are solubilized, and the protein may be refolded and / or cleaved by methods known in the art.

[0128] If the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a nucleotide sequence encoding a signal peptide, a stop codon, and a polyadenylation sequence. Optionally, the vector or gene construct may contain enhancers and introns. The gene construct can be introduced into eukaryotic host cells using conventional techniques. In some embodiments, the engineered gene in the expression vector encodes a signal peptide fused to the N-terminus of the protein to be produced. The secretion signal is typically cleaved when the protein is transported to the extracellular space, resulting in a mature protein secreted to the culture medium. In some embodiments, the signal peptide is a native signal peptide of human CD48, e.g., having the amino acid sequence of MCSRGWDSCLALELLLLPLSLLVTSIQG (SEQ ID NO: 10). In other embodiments, the signal peptide comprises a signal sequence from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, or prolactin.

[0129] To express a CD48 IgV domain variant, a CD48 fragment, a CD48-Fc fusion polypeptide, or a homomultimer (e.g., homodimer) thereof, the host cell can be transfected with a single expression vector. In some embodiments involving expression of a protein having two or more different polypeptides, a host cell can co-transfected with more than one expression vector. The expression vectors can be stably inserted into the genome of eukaryotic cells, resulting in stably transfected cells that can be used for protein production over more passages than transiently transfected cells. The stable transfection allows isolation of single clones of host cells that produce the recombinant protein at high yield. Following transfection, single clones can be isolated and high-yield clones selected using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.Attorney Docket No: IMID-102WO

[0130] The protein can be produced by growing (culturing) a host cell transfected with an expression vector under conditions that permit expression of the polypeptide(s) of the protein. For example, high-yield clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the proteins of the present disclosure. Following expression, the proteins can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification (e.g., for binding to an IgG Fc region or an affinity tag such as glutathione-S-transferase (GST) or histidine tag fused to the protein), gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography .IV. Pharmaceutical Compositions

[0131] The present disclosure also provides pharmaceutical compositions or formulations that contain a CD48 IgV domain variant, CD48 fragment, or CD48-Fc fusion protein described herein. The pharmaceutical composition can be formulated for use in a variety of drug delivery systems. One or more pharmaceutically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020).

[0132] In certain embodiments, a pharmaceutical composition may contain formulation materials 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. In such embodiments, suitable formulation materials 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-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); 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,Attorney Docket No: IMID-102WOthimerosal, 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, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see, Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020)).

[0133] Pharmaceutical compositions containing a CD48 IgV domain variant, CD48 fragment, or CD48-Fc fusion protein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous, subcutaneous, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0134] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof. An intravenous drug delivery formulation of the present disclosure may be contained in a bag, a pen, or a syringe. In certain embodiments, the bag may be connected to a channel including a tube and / or a needle.

[0135] In certain embodiments, the formulation is a liquid formulation. In certain embodiments, an aqueous formulation is prepared including the CD48 IgV domain variant, CD48 fragment, or CD48-Fc fusion protein in a pH-buffered solution. The pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and / or base. InAttorney Docket No: IMID-102WOcertain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide. In certain embodiments, a salt or buffer components may be added in an amount of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion.Intravenous formulations can be diluted with 0.9% sodium chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0136] In certain embodiments, the formulation is a lyophilized formulation including the CD48 IgV domain variant, CD48 fragment, or CD48-Fc fusion protein and a lyoprotectant. The lyoprotectant may be sugar, e.g., disaccharides. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and / or a preservative. The amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1:2 protein to sucrose or maltose. In certain embodiments, the protein to sucrose or maltose weight ratio may be of from 1 :2 to 1 :5. Before lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted between 6 to 8. In certain embodiments, the pH range for the lyophilized drug product may be from 7 to 8. In certain embodiments, a “bulking agent” may be added. A “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulations of the present disclosure may contain such bulking agents.

[0137] In certain embodiments, the lyophilized drug product may be constituted with an aqueous carrier. The aqueous carrier of interest herein is one which is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization. Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer’s solution or dextrose solution. In certain embodiments, the lyophilized protein product of the instant disclosure isAttorney Docket No: IMID-102WOconstituted to about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0138] The pharmaceutical compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents. The composition in solid form can also be packaged in a container for a flexible quantity.

[0139] In certain embodiments, the present disclosure provides a formulation with an extended shelf life including the CD48 Ig V domain variant, CD48 fragment, or CD48-Fc fusion protein, in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0140] A polyol, which acts as a tonicifier and may stabilize the antibody, may also be included in the formulation. The polyol is added to the formulation in an amount which may vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also be altered with respect to the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g., mannitol) may be added, compared to a disaccharide (such as trehalose). In certain embodiments, the polyol which may be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg / mL. In certain embodiments, the concentration of mannitol may be about 7.5 to about 15 mg / mL. In certain embodiments, the concentration of mannitol may be about 10 to about 14 mg / mL. In certain embodiments, the concentration of mannitol may be about 12 mg / mL. In certain embodiments, the polyol sorbitol may be included in the formulation.

[0141] A detergent or surfactant may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbates 20, 80 etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may includeAttorney Docket No: IMID-102WOa surfactant which is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4thed., 1996). In certain embodiments, the formulation may contain polysorbate 80 between about 0.1 mg / mL and about 10 mg / mL, or between about 0.5 mg / mL and about 5 mg / mL. In certain embodiments, about 0.1% polysorbate 80 may be added in the formulation.

[0142] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of elastomer complying with USP and Ph Eur. In certain embodiments, the liquid formulation may be diluted with 0.9% saline solution prior to use.

[0143] In certain embodiments, the liquid formulation of the disclosure may be prepared in combination with a sugar at stabilizing levels. In certain embodiments the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, a stabilizer may be added in an amount no greater than that which may result in a viscosity undesirable or unsuitable for intravenous administration. In certain embodiments, the sugar may be disaccharides, e.g., sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.

[0144] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multiuse (multiple-dose) formulation.

[0145] In certain embodiments, a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29).

[0146] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-Attorney Docket No: IMID-102WOglutamate, poly (2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.V. Therapeutic Uses

[0147] The CD48 IgV domain variants, CD48 fragments, and CD48-Fc fused proteins disclosed herein have improved immunotherapeutic properties as compared to a corresponding wild-type CD48 variant or protein. In certain embodiments, the CD48 IgV domain variant, CD48 fragment, or CD48-Fc fused protein has an increased binding affinity to CD2 compared to a corresponding wild-type CD48 variant or protein. In certain embodiments, the CD48 IgV domain variant, CD48 fragment, or CD48-Fc fused protein has an increased binding affinity to 2B4 compared to a corresponding wild-type CD48 variant or protein. In certain embodiments, the CD48 IgV domain variant, CD48 fragment, or CD48-Fc fused protein has increased binding affinities to both CD2 and 2B4 compared to a corresponding wild-type CD48 variant or protein. Such proteins are useful for reducing NK cell and T cell cytotoxicity.

[0148] Accordingly, the present disclosure also provides use of the CD48 IgV domain variant, CD48 fragment, or CD48-Fc fused protein to modulate the survival, proliferation, and / or activity of an immune cell. In certain embodiments, the present disclosure provides a method of modulating the interaction between an immune cell and a target cell, the method comprising contacting the immune cell and the target cell with a CD48 IgV domain variant, CD48 fragment, or CD48-Fc fused protein, or pharmaceutical composition or formulation disclosed herein. In certain embodiments, the present disclosure provides a method of modulating the survival, proliferation, and / or activity of an immune cell, the method comprising contacting the immune cell with a CD48 IgV domain variant, CD48 fragment, or CD48-Fc fused protein, or pharmaceutical composition or formulation disclosed herein. In certain embodiments, the immune cell is a T cell (e.g., a CD8+T cell, a CD4+T cell, a regulatory T cell, or an NKT cell) or an NK cell. In certain embodiments, the method reduces the cytotoxic activity of a CD8+T cell or an NK cell.

[0149] The present disclosure also provides use of the CD48 IgV domain variant, CD48 fragment, or CD48-Fc fused protein, or a pharmaceutical composition comprising any of them, in therapy, for example, as an immunosuppressive agent, e.g., for treating an autoimmune disease. In certain embodiments, the present disclosure provides a method of treating a disease or disorder associated with aberrantly increased immune activity (e.g.,Attorney Docket No: IMID-102WOlymphocyte activity), the method administering to a subject in need thereof an effective amount of the CD48 IgV domain variant, CD48 fragment, CD48-Fc fused protein, or pharmaceutical composition or formulation disclosed herein. The diseases and disorders suitable for such treatment include but are not limited to autoimmune diseases and inflammatory diseases. Examples of autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, psoriasis (e.g., psoriatic arthritis), celiac disease, graft-versus-host disease, myositis, and scleroderma. Examples of inflammatory diseases include gout, inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis), allergy, asthma, and transplantation. In certain embodiments, the CD48 IgV domain variant, CD48 fragment, CD48-Fc fused protein, or pharmaceutical composition or formulation disclosed herein is administered to the subject intravenously, e.g., by intravenous infusion.

[0150] The description above describes multiple aspects and embodiments of the disclosure. The patent application specifically contemplates all combinations and permutations of the aspects and embodiments.EXAMPLES

[0151] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.Example 1 - Preparation of exemplary CD48 IgV domains and variants fused with Fc domains

[0152] Human CD48 IgV domain variants were generated by creating single or multiple amino acid substitutions in the wild-type human CD48 IgV domain according to Table 1. Plasmids encoding the two Ig domains of wild-type human CD48 (NCBI reference NM_001778.3), with the indicated amino acid mutations in the IgV domain (Table 1), fused to the Fc region of human IgGl were constructed by site directed mutagenesis using splicing by overlap extension (SOE) PCR. Plasmid pCI-neo hCD48-Fc (see, Martinez-Vicente et al., (2019) PLOS PATHOG., 15 (4): el007658) was used as template and SOE PCR products were cloned into pCI-neo Fc vector as previously described (see, Perez-Carmona et al., (2015) J VIROL., 89 (22): 11323-11336). Control plasmid pCI-neo hCD58-Fc expressing the two Ig domains of human hCD58 fused to the Fc region of human IgGl was obtained by PCR using as template a construct encoding the corresponding full-length protein (NCBI reference NP_001770.1) and cloned into pCI-neo Fc vector. pCI-neoA43-Fc expressing the two Ig domains of the viral protein A43 fused to the Fc region of human IgGl was previouslyAttorney Docket No: IMID-102WOdescribed (see, Martinez-Vicente et al., (2019) PLOS PATHOG., 15 (4): el007658). pCI-neo sb2B4-Fc expressing the two Ig domains of a primate (Saiiiiiri boliviensis) 2B4 (“sb2B4”) fused to the Fc region of human IgGl, was used as a negative control, since this 2B4 protein does not interact with human proteins. Plasmids encoding the two Ig domains of hCD48, selected hCD48 IgV domain variants, hCD58 or sb2B4 fused to variants of human IgGl Fc domains were also employed, to abrogate the binding of the Fc region to Fc receptors. One Fc variant included mutations L234F, L235Q, K322Q, M252Y, S254T, and T256E see, Borrok et al., (2017) J PHARM Set., 106(4): 1008-1017), and the corresponding fusion proteins were indicated as Fc* proteins. Another Fc variant included mutations L234A, L235A, M252Y, S254T, and T256E (see, Sazuki et al., (2021) MAbs, 13: 1923366), and the corresponding fusion proteins were indicated as Fc#proteins.

[0153] Fusion proteins were produced by transiently transfecting HEK-293T cells. 5-7 days after transfection, the supernatants containing the fusion proteins were harvested and subsequently clarified to remove cellular debris. When necessary, the supernatants containing the Fc fusion proteins were concentrated using an Amicon Ultra- 15 centrifugal filter unit. The Fc fusion proteins were quantified by sandwich enzyme-linked immunosorbent assay (ELISA) using anti-human Fc IgG mAb and anti-human IgG (Fc specific) POD secondary antibody. Purified Fc, Fc*, and Fc#proteins were produced using CHO cell expression platform Turbo-CHO and their purity confirmed by SDS-PAGE and SECHPLC (>95%). Example 2 - Binding of exemplary CD48 IgV domain variant Fc-fusion proteins to CD2 and 2B4-expressing cells

[0154] This example demonstrates the ability of purified CD48 IgV domain variant Fc-fusion proteins, as produced according to the method described in Example 1, to bind cells expressing 2B4 or CD2 on the surface.

[0155] Each fusion protein comprising a hCD48 IgV domain variant was measured for binding to human 2B4 and human CD2 expressing cells by flow cytometry. Briefly, IxlO3YT cells, endogenously expressing h2B4, or Jurkat cells, which endogenously express hCD2, were incubated with 5 pg / mL of the Fc fusion proteins for 30 min at d’C, followed by incubation with the anti-human IgG (Fc specific) PE antibody. The hCD58-Fc fusion protein was used as a positive control for hCD2 binding, the viral protein A43-Fc fusion protein as a positive control for binding to h2B4 and in a lesser extent to hCD2, and the sb2B4-Fc fusion protein (also referred to as an “unrelated” Fc fusion protein herein) as a negative control. The stained cells were analyzed by flow cytometry.Attorney Docket No: IMID-102WO

[0156] hCD48-Fc variants containing a single residue substituted of wild-type human CD48 IgV domain (hCD48-Fc SI, hCD48-Fc S2, hCD48-Fc S3, and hCD48-Fc S4) were analyzed. As shown in FIGs. 2A-2C, hCD48-Fc S 1 , which incorporates the mutation Q27E, improved the binding to both h2B4 and hCD2 when compared to wild-type hCD48-Fc. hCD48-Fc S2, CD48-Fc S3, and CD48-Fc S4 did not show a significant enhanced ability to bind to hCD2-expressing cells, or h2B4-expressing cells as compared to wild-type hCD48-Fc at the concentration examined. As a positive control, hCD58-Fc showed a marked interaction with CD2-expressing cells. The control of the unrelated Fc fusion protein at the equivalent concentration did not yield substantial binding to cells expressing h2B4 or hCD2.

[0157] The binding of the hCD48-Fc variants bearing individual substitutions of adjacent residues in the IgV domain, namely, hCD48-Fc Al, hCD48-Fc A2, and hCD48-Fc A3, was assessed. As shown in FIGs. 3A-3B, hCD48-Fc Al, bearing the mutation S43V, R44K, K45R, and S46L, exhibited an improved binding only to h2B4-expressing cells when compared with wild-type hCD48-Fc at the concentration examined. hCD48-Fc A2 that contains the mutation N76A, S77N, and T78I led to an increased interaction with hCD2, although a reduced binding of this protein to h2B4 expressing cells was observed. Finally, hCD48-Fc A3 that incorporates the mutations G88M and N89F resulted in augmented levels of interaction with h2B4 and modestly to hCD2 as compared to wild-type hCD48-Fc.

[0158] As shown in FIGs.4A-4B, the binding of the hCD48-Fc variants containing a combination of single and adjacent residue substitutions of wild-type human CD48 IgV domain, hCD48-Fc Cl, hCD48-Fc C2, hCD48-Fc C3, hCD48-Fc C4, and hCD48-Fc C5, was evaluated. All the variants tested yielded a striking increased interaction with hCD2 expressing cells and augmented binding of varying degrees to h2B4-expressing cells as compared to wild-type hCD48-Fc. Among the five combination domain variants analyzed, hCD48-Fc C2 comprising mutations Q27E / F34Y / N76A / S77N / T78I / G88M / N89F, hCD48-Fc C4 comprising mutations Q27E / F34Y / N76A / S77N / T78I / K86D / G88M / N89F, and hCD48-Fc C5 comprising mutations Q27E / F34Y / S43V / R44K / K45R / S46L / N76A / S77N / T78I / K86D / G88M / N89F displayed the highest levels of interaction to h2B4 and hCD2 at the concentration examined.

[0159] The interaction levels of the wild-type hCD48-Fc variants to 2B4-expressing cells and CD2-expressing cells examined for hCD48-Fc SI, hCD48-Fc S2, CD48-Fc S3, hCD48-Fc Al, hCD48-Fc A2, hCD48-Fc A3, hCD48-Fc Cl, hCD48-Fc C2, hCD48-Fc C3, hCD48-Fc C4, and hCD48-Fc C5 with h2B4 and hCD2 are summarized in Table 6. ThisAttorney Docket No: IMID-102WOtable shows the fold-change of binding (MFI) of each hCD48-Fc mutant relative to the MFI of the wild-type hCD48-Fc.Table 6: Binding of CD48 IgV domain variants to 2B4- and CD2-expressing cells

[0160] The binding of hCD48-Fc SI, hCD48-Fc Cl, hCD48-Fc C4 and hCD48-Fc C5 variants to COS-7 cells transiently expressing h2B4 and hCD2, was measured by flow cytometry. Briefly, IxlO5COS-7 cells transiently transfected with the pCI-neo h2B4 plasmid or the pCI-neo hCD2 plasmid, were stained with 5 |lg / mL of CD48 IgV domain variant Fc-fusion proteins or control Fc fusion proteins for 30 min at 4°C, followed by incubation with the anti-human IgG (Fc specific) PE. The hCD58-Fc fusion protein was used as a control for hCD2 binding, and the unrelated sb2B4-Fc fusion protein as a negative control. The stained cells were analyzed by flow cytometry.

[0161] As shown in FIGs.5A-5B, hCD48-Fc SI , hCD48-Fc Cl , hCD48-Fc C4 and hCD48-Fc C5 variants displayed increased binding to COS-7 cells expressing h2B4 and hCD2, as compared to wild-type hCD48-Fc, whereas no interaction of these proteins with non-transfected COS-7 cells was observed. hCD48-Fc C5 was the hCD48-Fc variant reaching the highest binding levels to both h2B4 and hCD2. As a positive control, hCD58-FcAttorney Docket No: IMID-102WOshowed a marked interaction with hCD2-expressing cells. The control of the unrelated Fc fusion protein at the equivalent concentration did not reached significant binding to COS-7 cells expressing h2B4 or hCD2.

[0162] The strength of the binding of the hCD48-Fc variants, hCD48-Fc S I, hCD48-Fc Cl , hCD48-Fc C4, and hCD48-Fc C5, in parallel to wild type hCD48-Fc and the unrelated sb2B4-Fc fusion protein, to YT and Jurkat cells was examined by flow cytometry performing a dose response of the purified Fc fusion proteins.

[0163] As shown in FIG. 6A, the four hCD48-Fc variants displayed substantially increased levels of interaction with YT cells as compared to wild-type hCD48-Fc, with hCD48-Fc C5 being the variant showing the highest binding, followed by hCD48-Fc C4, and then the hCD48-Fc Cl and hCD48-Fc SI variants. ECso values for wild-type hCD48-Fc, hCD48-Fc C4, and hCD48-Fc C5 were 33.6, 1.21, and 0.16 pg / mL, respectively. A significant binding of the unrelated Fc fusion protein to YT cell was not detected. As shown in FIG. 6B, similar result was obtained when the binding of the hCD48-Fc variants to Jurkat cells was assessed, although in this case a significant interaction of hCD48-Fc SI was not appreciated. ECso values for hCD48-Fc C4 and hCD48-Fc C5 were 3.9 and 1.99 pg / mL, respectively.

[0164] To gain further understanding of the molecular and atomic details of hCD48-Fc C5 recognition of 2B4 and CD2, structural models of hCD48-Fc C5:2B4 and hCD48-Fc C5:CD2 complexes were derived. Overall, the structural arrangement of the hCD48-Fc C5 mutant in complex with h2B4 and hCD2 was very similar, with both ligands interacting mainly by stacking of beta strands in the central region of the interface (core) and by loops in peripheral regions (rim). Besides Q27E, which is located in strand C and in the central region of the interaction, the remaining changes were positioned in the rim of the interface. In terms of interface area, both complexes were comparable, with hCD48-Fc C5:h2B4 and hCD48-Fc C5:hCD2 having an interface contact area of 1554 Ang2and 1602 Ang2respectively (data not shown).

[0165] Having the structure of the protein complexes also allowed understanding of the contribution of the different substitutions in hCD48-Fc C5 to the binding. The twelve mutations introduced in hCD48-Fc C5 contributed in very different ways to the interaction with h2B4, hCD2, or both (data not shown). Among the single residue replacements, Q27E and F34Y mainly contributed to non-bonded polar interactions with 2-3 residues of eitherAttorney Docket No: IMID-102WOpartner. In contrast, K86D was not directly located at the interface and therefore did not interact with either h2B4 or hCD2.

[0166] Within the set of changes involving short stretches of adjacent residues, G88M and N89F in positions 88 and 89 interacted extensively with h2B4 and hCD2, contributing with atomic interactions with 5 and 4 different residues in the corresponding partners, respectively. Residues 43 to 46 located in the C'C" loop, encompassing the changes S43V, R44K, K45R, and S46L, participated in the interaction with residue K76 in hCD2, but no interactions were observed with h2B4 as the loops were positioned further away from the interface compared to hCD2 (data not shown). Likewise, residues 76 to 78, which correspond to a glycosylation site immediately before 0 strand F in the changes N76A, S77N, and T78I, were also located in a region that was not part of the interface and therefore did not contribute to the interaction with any of the two partners.

[0167] After identifying the atomic details of the hCD48-Fc C5 interactions with h2B4 and hCD2, the affinity of the complexes was quantified by predicting binding energies from the structural models and compared with those of wild-type hCD48 with both partners. The hCD48-Fc C5:h2B4 complex exhibited a substantially lower binding energy than the hCD48:h2B4 complex, indicating a stronger interaction (data not shown). Likewise, the hCD48-Fc C5:hCD2 complex displayed a markedly reduced binding energy relative to the hCD48:hCD2 complex (data not shown). These findings were consistent with the binding data obtained by flow cytometry and ELISA. Together, they indicated that the amino acid substitutions in hCD48-Fc C5 markedly enhanced its affinity for both h2B4 and hCD2. Example 3 - Binding of exemplary CD48 IgV domain variant Fc-Fusion Proteins to soluble CD2 and 2B4 by ELISA and SPR

[0168] This example demonstrates the capacity of purified hCD48-Fc SI, hCD48-Fc Cl, hCD48-Fc C4, and hCD48-Fc C5 variants, as produced according to the method described in Example 1 , to bind to soluble 2B4 and CD2 by ELISA and SPR.

[0169] The potency of the binding of the hCD48-Fc variants, hCD48-Fc SI, hCD48-Fc CL hCD48-Fc C4, and hCD48-Fc C5, in parallel to wild type hCD48-Fc and the unrelated sb2B4-Fc fusion protein used as a negative control, to h2B4 and hCD2 was also analyzed by ELISA performing a dose response of the Fc fusion proteins. The hCD58-Fc fusion protein was used as a control for hCD2 binding. Plates coated with 2 pg of immobilized h2B4-His or hCD2-His were incubated with different concentrations of the purified Fc fusion proteins, and analyzed by ELISA using the anti -human IgG (Fc-specific) POD antibody.Attorney Docket No: IMID-102WO

[0170] As shown in FIGs.7A-7B, the results obtained with the four hCD48-Fc variants by ELISA, when h2B4 and hCD2 were used to coat the plates, followed a trend comparable to that obtained by flow cytometry. hCD48-Fc C5 showed the most elevated level of interaction with both proteins (ECso values of 0.06 pg / mL for h2B4 and 13.8 pg / mL for hCD2), followed by hCD48-Fc C4 (ECso values of 0.12 pg / mL for h2B4 and 35.7 pg / mL for hCD2), and then hCD48-Fc Cl, and in all cases the interaction displayed was superior to that of wild type hCD48-Fc (ECso values of 2.6 pg / mL for h2B4). hCD48-Fc SI was capable to bind to almost the same extent than hCD48-Fc Cl to h2B4, but a significant interaction of this hCD48-Fc variant to hCD2 was not observed.

[0171] The binding of purified wild-type hCD48-Fc and hCD48-Fc C5 to h2B4 and hCD2 was further analyzed by surface plasmon resonance (SPR) kinetic analyses, employing different concentrations of Fc-fusion proteins and recombinant h2B4 or hCD2 proteins immobilized onto sensor chips. The hCD58-Fc fusion protein was used as a control for hCD2 binding. Recombinant His-tagged h2B4 and hCD2 proteins were immobilized onto a Series S sensor chip CM5 via anti-histidine antibodies and increasing concentrations of recombinant Fc fusion proteins were injected in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% vol / vol surfactant P20, pH 7.4). Sensor chip surfaces were regenerated with 10 mM glycine-HCl [pH 1.5]. Kinetic and affinity constants were calculated using Biacore Insight Evaluation Software and sensorgrams were fitted to a 1:1 binding model.

[0172] As shown in Table 7, the hCD48-Fc C5 fusion protein demonstrated markedly improved binding to h2B4 relative to wild-type hCD48-Fc to h2B4. The equilibrium dissociation constants (KD) exhibited were: 2.62 nM for hCD48-Fc, and 0.243 nM for hCD48-Fc C5. No binding of hCD58-Fc to h2B4 was detected. In the case of the interaction with hCD2, the KD value determined for hCD48-Fc C5 was 62.6 nM, whereas the binding of hCD48-Fc was not detected. The KD of the binding of hCD58-Fc to hCD2 was 23.6 nM. These results provide additional support for interaction data obtained by ELISA.Table 7: Binding affinities (KD) of IgV domain variants to h2B4 and hDC2Attorney Docket No: IMID-102WOExample 4 - Competition of the binding of CD48 to 2B4 and CD58 to CD2 by exemplary CD48 IgV domain variant Fc-Fusion Proteins

[0173] This example demonstrates the capacity of purified hCD48-Fc Cl, hCD48-Fc C4, and hCD48-Fc C5 variants, as produced according to the method described in Example 1, to modulate the binding of CD48 to 2B4 and CD58 to CD2 using both cell-based flow cytometry assays and solution-based ELISA assay.

[0174] The ability of the hCD48-Fc variants, hCD48-Fc Cl , hCD48-Fc C4, and hCD48- Fc C5, in parallel to wild type hCD48-Fc, hCD58-Fc and the unrelated sb2B4-Fc fusion protein, to block the binding of hCD48 to h2B4 and hCD58 to hCD2 was evaluated. Plates coated with 2 pg of immobilized h2B4-His or hCD2-His were incubated with different concentrations of purified hCD48-Fc variants, wild type hCD48-Fc, hCD58-Fc, or the unrelated control fusion protein, followed by the ligand hCD48-murine Fc (in the case of hCD48-h2B4 binding) or hCD58-murine Fc (in the case of hCD58-hCD2 binding), and analyzed by ELISA using an anti-mouse Fc-POD.

[0175] As shown in FIG.8A, the three hCD48-Fc variants, at the different concentrations tested, inhibited the interaction between hCD48 and h2B4 more efficiently than wild-type hCD48-Fc. The blockage of the hCD48-h2B4 binding displayed by hCD48-Fc C4 and hCD48-Fc C5 was higher than that of hCD48-Fc Cl . In all cases, neither the unrelated Fc fusion protein nor hCD58-Fc significantly affected the interaction. As shown in FIG. 8B, hCD48-Fc C4, hCD48-Fc C5, and in a lesser extent hCD48-Fc Cl , blocked in a dose responsive manner the hCD58-hCD2 interaction, whereas wild-type hCD48-Fc or the unrelated Fc fusion protein did not substantially alter it. As a positive control, hCD58-Fc showed a dose-response effect competing the binding of hCD58 to hCD2. Therefore, hCD48-Fc C4, hCD48-Fc C5, and in a lesser extent hCD48-Fc Cl, have the capacity to block the interaction between hCD48 and h2B4 and hCD58 and hCD2.

[0176] The ability of hCD48-Fc C5 to compete with the interactions between CD48 and CD58 and their natural ligands was analyzed using ligands expressed at the cell surface. HEK-h2B4 transfected cells or Jurkat cells were incubated with varying concentrations of purified Fc-fusion proteins (0.1, 0.5, and 5 pg / mL for HEK-h2B4 transfected cells: 1, 5, 10 pg / mL for Jurkat cells), followed by another incubation with 3.5 or 5.5 pg / ml of CD48-murine Fc (CD48-mFc) for HEK h2B4 cells or CD58-murine Fc (CD58-mFc) for Jurkat cells, respectively. The CD48-mFc and CD58-mFc fusion proteins consisted of the ectodomains of CD48 or CD58 linked to the Fc portion of murine IgGl. Detection wasAttorney Docket No: IMID-102WOperformed with an anti-mouse IgG PE (Fc specific) antibody. Cells were analyzed by flow cytometry.

[0177] Results showed a strong dose response inhibition of both hCD48:h2B4 and hCD58:hCD2 interactions by the hCD48-Fc C5 mutant (FIGs. 8C and 8D). Importantly, the blocking capacity of hCD48-Fc C5 was much more potent than that of the wild-type hCD48-Fc. In contrast, the binding of hCD48 to h2B4 or hCD58 to hCD2 was unaltered after treatment with the unrelated Fc control protein. Interestingly, the ability of mutant C5-Fc to block the interaction between hCD58 and hCD2 was much more pronounced compared to the hCD58-Fc protein.Example 5 - Inhibition of NK and T cell conjugate formation by exemplary CD48 IgV domain variant Fc-Fusion Proteins

[0178] This example demonstrates the ability of purified hCD48-Fc Cl, hCD48-Fc C4, and hCD48-Fc C5 variants, as produced according to the method described in Example 1, to modulate the formation of conjugates between NK or T cells and target cells.

[0179] To assess the impact of hCD48-Fc Cl, hCD48-Fc C4, and hCD48-Fc C5 variants on cell conjugate mediated by hCD48-h2B4 interaction, YT cells, endogenously expressing h2B4, and target HEK cells transiently expressing hCD48 were separately labeled with two different fluorescent dyes for 20 min at 37°C followed by washing. Then, 1 x lCP labeled YT cells were co-cultured with labeled HEK cells expressing hCD48 at a 1 / 1 ratio in a final volume of 150 pL in the absence or presence of soluble Fc fusion proteins, for 0 or 20 min at 37°C. Final concentrations of the Fc fusion proteins in the co-cultures were 5 pg / mL.Reactions were stopped by adding 50 pL of ice-cold PBS. Conjugates were detected by flow cytometry as double positive events. The percentage of conjugates formed at 20 min was determined after substracting conjugates formed by untransfected HEK cells. Alternatively, CFSE-labeled hCD48-stably transfected K562 cells were incubated at 37°C with YT cells stained with CMAC at a 1 / 1 ratio, in the absence (Ctl+) or presence of 5 pg / mL of purified hCD48-Fc#, hCD48-Fc#C5, or an unrelated control Fc#protein (Ctl-Fc#), and the formation of conjugates examined after 0 and 20 min. Conjugates were identified as double positive cells by flow cytometry. The percentage of conjugates formed at 20 min was determined after substracting conjugates formed by untransfected K562 cells.

[0180] As shown in FIG. 9A hCD48-Fc Cl , hCD48-Fc C4 and hCD48-Fc C5 variants inhibited the formation of conjugates mediated by hCD48 and h2B4 more efficiently thanAttorney Docket No: IMID-102WOwild-type hCD48-Fc, with hCD48-Fc C5 displaying the higher blocking capacity. The unrelated Fc fusion protein was unable to significantly affect this process. FIG. 9C also shows an inhibition of conjugate formation between YT cells and CD48-expressing K562 cells in the presence of hCD48-Fc#C5, as compared to co-cultures non-exposed to Fc#fusion proteins or incubated with wild-type CD48-Fc#. These results demonstrated that the hCD48-Fc C4, hCD48-Fc C5, hCD48-Fc#C5 variants, and in a lesser extent the hCD48-Fc Cl variant, were effective at blocking the formation of NK-cell conjugates and target cells mediated through the interaction between hCD48 and h2B4.

[0181] To examine the impact of the hCD48-Fc Cl, hCD48-Fc C4, and hCD48-Fc C5 variants on cell conjugate mediated by hCD58-hCD2 interaction, Jurkat cells endogenously expressing hCD2, and target K562 cells endogenously expressing hCD58, were separately labeled with different fluorescent dyes, for 20 min at 37° C followed by washing. Then, lx 10’ labeled Jurkat cells were co-cultured with K562 cells at a 1 / 1 ratio in a final volume of 150 pL in the absence or presence of Fc* fusion proteins containing mutations in the Fc region that abrogates the binding of the Fc region to Fc receptors, for 0 or 20 min at 37°C. Final concentrations of the Fc* fusion proteins in the co-cultures were 5 pg / mL. Reactions were stopped by adding 50 pL of ice-cold PBS. Conjugates were detected by flow cytometry as double positive events.

[0182] As shown in FIG. 9B, hCD48-Fc* C4 and hCD48-Fc* C5 variants significantly interfered with the formation of conjugates mediated by hCD58 and hCD2 interaction, whereas wild-type hCD48-Fc* or the unrelated Fc* protein did not substantially alter conjugate formation. hCD48-Fc* Cl barely interfere with this process at the concentration tested. As a positive control, the hCD58-Fc* diminished the proportion of conjugates. These results demonstrated that the hCD48-Fc* C4, and hCD48-Fc* C5 variants were effective blocking the formation of T-cell conjugates with target cells mediated through the interaction between hCD58 and hCD2.Example 6 - Inhibition of 2B4- or CD2-mediated NK cell cytotoxicity by an exemplary CD48 IgV domain variant

[0183] This example demonstrates the ability of the purified hCD48-Fc C5 variant, as produced according to the method described in Example 1 , to modulate 2B4- and CD2-dependent NK cell cytotoxicity against target cells.

[0184] To assess the ability of the hCD48 Fc C5 variant to inhibit the cytotoxicity of 2B4-expressing cells, the cytotoxic activity of YT effector cells was determined in a calcein-Attorney Docket No: IMID-102WOAM release assay (see, Neri et al., CLIN. DIAGN. LAB. IMMUNOL. (2001) 8:1131-1135). The assay was performed in V bottom 96-well microtiter plates. Briefly, target HEK or HEK transiently transfected with hCD48 were labeled with calcein at 2 pM for 30 min at 37°C. After two washes, 5xl03target labeled cells per well were mixed at 37°C with effector cells that had been pre -incubated with purified Fc fusion proteins for 30 min at 37°C at E / T ratios of 5 / 1, or 10 / 1. Final concentrations of the Fc fusion proteins in the co-cultures were 5 pg / mL. The assay also included wells with only target cells (spontaneous release), only target cells lysed with the addition of 1% Triton X-100 (maximum release), or medium alone (background). After four hours, plates were centrifuged at 1600 rpm for 6 min at 4°C, and 100 uL of supernatant of each culture was collected and transferred into new plates.Fluorescence was measured using a multimode microplate reader. The background fluorescence was subtracted from all samples. The percentage of specific lysis was calculated according to the formula: (experimental release — spontaneous release) / (maximum release — spontaneous release) x 100.

[0185] As shown in FIGs. 10A and 10B, the killing displayed by the YT cells toward untransfected HEK (FIG. 10A) or K562 (FIG. 10B) cells, which do not constitutively express hCD48, was low. In contrast, the expression of hCD48 in the HEK or K562 cells transiently transfected with this receptor substantially enhanced the cytotoxicity of the YT cells against these cells at the two different ratios evaluated. hCD48-Fc C5 fusion protein led to a significant reduction of YT cell-mediated cytotoxicity towards the hCD48 expressing target cells as compared to that caused by wild-type hCD48-Fc and the irrelevant Fc fusion protein. Hence, mutations introduced in the IgV domain of the soluble hCD48-Fc molecule that give rise to the hCD48-Fc C5 variant increase its capacity to suppress 2B4-dependent NK cell cytotoxicity.

[0186] To assess the ability of the hCD48 C5 variant to inhibit the cytotoxicity of hCD2-expressing cells, the cytotoxic activity of NK-92 effector cells, which endogenously express this receptor, was determined in a calcein- AM release assay (see Neri et al., CLIN. DIAGN. LAB. IMMUNOL.(2001) 8:1131-1135). The assay was performed in V bottom 96-well microtiter plates. Briefly, target K562 were labeled with calcein at 2 pM for 30 min at 37°C. After two washes, IxlO4target labeled cells per well were mixed at 37°C with effector cells that had been pre -incubated with Fc#proteins containing the version of the Fc region that abrogates the binding of the Fc region to Fc receptors for 30 min at 37°C, at an E / T ratio of 0.5 / 1 and 2 / 1. Final concentrations of the Fc#fusion proteins in the co-cultures were 5 pg / mL. The assay also included wells with only target cells (spontaneous release), only target cellsAttorney Docket No: IMID-102WOlysed with the addition of 1% Triton X-100 (maximum release), or medium alone (background). After four hours, plates were centrifuged at 1600 rpm for 6 min at 4°C, and 100 uL of supernatant of each culture was collected and transferred into new plates.Fluorescence was measured using a multimode microplate reader. The background fluorescence was subtracted from all samples. The percentage of specific lysis was calculated according to the formula: (experimental release — spontaneous release) / (maximum release — spontaneous release) x 100.

[0187] As shown in FIG. 11, hCD48-Fc#C5 resulted in a significant reduction of the cytotoxicity displayed by NK-92 cells against K562 cells, which was not observed when the effector cells were incubated with a similar concentration of wild-type hCD48-Fc#or an irrelevant Fc#protein. As a positive control, hCD58-Fc#led to a decrease of NK-92 cell-mediated cytotoxicity towards K562 cells. Therefore, the mutations present in the IgV domain of the hCD48-Fc#C5 variant enable this molecule to inhibit hCD2-dependent NK cell cytotoxicity.Example 7 - hCD48-Fc C5-Fc#reduces cytokine production and T cell proliferation in a mixed lymphocyte reaction assay

[0188] This example demonstrates the ability of the purified hCD48-Fc#C5 variant, as produced according to the method described in Example 1, to modulate T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay.

[0189] To evaluate the potential impact of hCD48-Fc#C5 on T cell responses, a one-way MLR assay was performed. PBMCs isolated from buffy coats of healthy donors were used in different pairwise combinations. Irradiated (30 Gy) PBMCs from one donor were employed as stimulator cells and PBMCs from a second donor, stained with 2.5 pM CellTrace CFSE, were employed as responder cells, lx IO3stimulator cells were incubated with responder cells at a 1 / 1 E / T ratio per well in round-bottom 96-well culture plates, in the absence or presence of 5 pg / mL of Fc#purified fusion proteins, at 37°C for 6 days. At the end of this period, culture supernatants and cells were collected. Levels of IFN-y and TNF-a in cell-free culture supernatants were determined by ELISA. Proliferation of responder PBMCs was determined based on CFSE signal dilution by flow cytometry. The proliferation of responder CD4+and CD8+T cells were assessed by staining with anti-CD4 PE and anti-CD8 Alexa Fluor 647.

[0190] As shown in FIG. 12A, although donor-to-donor variability was observed, T cell proliferation was consistently reduced in the presence of hCD48-Fc#C5. Similar effects were seen for both CD4+and CD8+T cell subsets (FIGs. 12B and 12C). Notably, hCD48-Fc#C5Attorney Docket No: IMID-102WOpresented a higher inhibition than hCD58-Fc#. Assessment of cytokine secretion revealed that hCD48-Fc'' C5 demonstrated marked suppressive efficacy, with a substantial decrease of both IFN-y and TNF-a, in particular IFN-y, across all donor pairs evaluated (FIGs. 12D and 12E).Conversely, the amount of IFN-y and TNF-a released was not significantly affected by the unrelated control Fc#protein (Ctl-Fc#). Hence, the mutations present in the IgV domain of the hCD48-Fc#C5 variant enable this molecule to inhibit T cell proliferation and IFN-y and TNF-a production in MLR assays.Example 8 - Identification of two potential immunogenic hot spots in the hCD48-Fc C5-Fc#mutant and their binding properties

[0191] This example demonstrates the computational evaluation of potential immunogenic hot spots in the hCD48-Fc#C5 variant, and the generation and characterization of derivative variants with reduced immunogenicity.

[0192] The prediction of immunogenicity in the different sequences was performed using NetMHCIIpan version 4.3 (Nilsson et al. 2023), a machine -learning prediction method based on Neural Network models trained using MHC-II immunopeptidomics. Each sequence variant was scanned against a set of 49 HLA-II as overlapping 13 to 19-mer peptides to compute an immunogenicity score at a given residue position. The score of a given peptide was considered a potential epitope if the predicted rank value was less than or equal to 1 % as previously reported (Reynisson et al. 2020; Attermann et al. 2021). Binding assays were performed by flow cytometry as described in Example 2.

[0193] As shown in FIG. 13, the prediction identified two potential immunogenic hot spots in hCD48-Fc#C5 when self-adjusted scoring, which accounts for peptides present in the human proteome, was applied. These were the stretches around residues 27-41 (YTYDQKIVE (SEQ ID NO: 49), which includes the substitution F34Y) and around residues 78-89 (IYIMRVLKD (SEQ ID NO: 50), encompassing the replacement K86D). Additional peaks of immunogenicity were detected between residues 100-120 and within regions 240-280 and 370-410, corresponding to the fused Fc#domain. However, when the scores were adjusted for self-tolerance, the risk score for all three regions was reduced to zero. The immunogenic liabilities of hCD48-Fc" C5 were further reflected in the overall non-adjusted and self-adjusted scores, which were 3.175386 and 0.4189657, respectively (Table 8).

[0194] Taking into consideration the two potential immunogenic hot spots of hCD48-Fc#C5, three new hCD48-Fc#variants were generated by reverting residues Y34, D86, or both residues simultaneously to amino acids F34 and K86 present in wild-type hCD48. The three new hCD48-Fc#C5 mutants are named hCD48-Fc#mH (reverting residue Y34 to wild-typeAttorney Docket No: IMID-102WOF34 in hCD48-Fc#C5), hCD48-Fc#mI (reverting residue D86 to wild-type K86 in hCD48-Fc" C5), and hCD48-Fc" R1 (reverting both residues Y34 and D86 to wild-type F34 and K86 in hCD48-Fc#C5). The predicted immunogenic profiles indicated that these hCD48-Fc#C5-derived variants exhibited reduced immunogenicity, particularly in the case of hCD48-Fc#Rl where the self-adjusted risk score dropped to 0 due to the removal of both immunogenic hot spots (Table 8). Additionally, mutants hCD48-Fc#mH and hCD48-Fc#mI also showed improved immunogenic profiles (Table 8).Table 8: The non-adjusted and self-adjusted risk scores for each of the mutants

[0195] To evaluate the binding properties of the three newly generated hCD48-Fc" mutants, the ability of different concentrations of hCD48-Fc#mH, hCD48-Fc#mI, and hCD48-Fc#R1 to interact with YT cells and Jurkat cells was analyzed by flow cytometry, comparing their interactions to those of hCD48-Fc#C5. As illustrated in FIG. 14A, hCD48-Fc" mH and hCD48-Fc#mI, which incorporated back mutations in only one of the two potential hot spots, demonstrated binding to h2B4-expressing cells at levels comparable to hCD48-Fc#C5, particularly hCD48-Fc#ml. In contrast, the hCD48-Fc#R1 mutant, in which both potential hot spots were removed, displayed decreased interaction with this receptor across all tested concentrations. In assays with Jurkat cells (FIG. 14B), hCD48-Fc#Rl also exhibited a substantial loss of interaction relative to hCD48-Fc#05, hCD48-Fc#mH showed a lesser decrease, while hCD48-Fc#mI maintained binding levels approaching those of hCD48-Fc#C5. The unrelated Fc#fusion protein at equivalent concentrations did not yield substantial binding to cells expressing h2B4or CD2, confirming the specificity of the interactions.Therefore, while reverting the aspartic acid at position 86 in hCD48-Fc#C5 to the native lysine found in hCD48 largely maintained binding to h2B4 and CD2, substituting the tyrosine at position 34 with phenylalanine, and especially restoring both potential immunogenic hot spots to the hCD48 sequence, resulted in partial reduction in binding to both receptors.Attorney Docket No: IMID-102WOExample 9 - Functional analysis of soluble C5-Fc#-derived mutants devoid of potential immunogenic hot spots

[0196] This example demonstrates the ability of hCD48-Fc#variants hCD48-Fc" mH, hCD48-Fc#ml, and hCD48-Fc#Rl, in which one or both potential immunogenic hot spots were eliminated, to modulate T cell proliferation and cytokine production in MLR assays.

[0197] To determine whether soluble hCD48-Fc#mH, hCD48-Fc#ml, and hCD48-Fc#Rl were functionally competent, their ability to alter T cell proliferation and IFN-y production in MLR assays was examined. MLR assays were performed as described in Example 7. Briefly, CFSE-labeled responder PBLs were co-cultured with irradiated allogeneic stimulator PBLs at a 1 / 1 ratio for 6 days in the absence or presence of 5 pg / mL of Fc#purified fusion proteins at 37°C. T cell proliferation was determined based on CFSE signal dilution by flow cytometry, and levels of IFN-y in cell-free culture supernatants were determined by ELISA.

[0198] As illustrated in FIG. 15A, decreased T cell proliferation was observed in the presence of hCD48-Fc#ml, to a comparable extent to that exhibited by hCD48-Fc#C5. In line with the binding assays, the impact of hCD48-Fc#mH and hCD48-Fc#Rlwas lower than that of hCD48-Fc#C5. An expanded set of donor pairs was then used to assess the effects of the hCD48-Fc#mutants on IFN-y secretion in the MLR co-cultures. Consistent with the proliferation data, mI-Fc#substantially reduced IFN-y production, whereas hCD48-Fc#mH and hCD48-Fc#Rl exhibited more modest effects (FIG. 15B). In contrast, IFN-y levels were not substantially altered in the presence of the unrelated control protein (Ctl-Fc#). Thus, in addition to hCD48-Fc" C5, hCD48-Fc#ml also attenuates T cell proliferation and IFN-y production in MLR assays.INCORPORATION BY REFERENCE

[0199] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0200] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

Attorney Docket No: IMID-102WOCLAIMS1. A CD48 IgV domain variant comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1 and / or at least 80% identical to SEQ ID NO: 15, wherein the CD48 IgV domain variant comprises one or more mutations at one or more amino acid positions selected from 27, 34, 43-46, 76-78, 86, 88, and 89, corresponding to SEQ ID NO: 1.

2. The CD48 IgV domain variant of claim 1, wherein the CD48 IgV domain variant does not contain just a single point mutation of a Tyr (Y) for Gin (Q) at amino acid position 27.

3. The CD48 IgV domain variant of claim 1 or 2, comprising one or more of: Glu (E) at amino acid position 27, Tyr (Y) at amino acid position 34, Vai (V) at amino acid position 43, Lys (K) at amino acid position 44, Arg (R) at amino acid position 45, Leu (L) at amino acid position 46, Ala (A) at amino acid position 76, Asn (N) at amino acid position 77, He (I) at amino acid position 78, Asp (D) at amino acid position 86, Met (M) at amino acid position 88, and Phe (F) at amino acid position 89, corresponding to SEQ ID NO: 1.

4. The CD48 IgV domain variant of any one of claims 1-3, comprising one or more of: (a) Glu (E) at amino acid position 27;(b) Tyr (Y) at amino acid position 34;(c) Vai (V) at amino acid position 43, Lys (K) at amino acid position 44, Arg (R) at amino acid position 45, and Leu (L) at amino acid position 46;(d) Ala (A) at amino acid position 76, Asn (N) at amino acid position 77, and He (I) at amino acid position 78;(e) Asp (D) at amino acid position 86; and(f) Met (M) at amino acid position 88, and Phe (F) at amino acid position 89.

5. The CD48 IgV domain variant of claim 4, comprising the mutation of (a).

6. The CD48 IgV domain variant of claim 4 or 5, comprising the mutations of (d).

7. The CD48 IgV domain variant of any one of claims 4-6, comprising (a), (d), and (f).

8. The CD48 IgV domain variant of claim 7, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 11.Attorney Docket No: IMID-102WO9. The CD48 IgV domain variant of claim 8, comprising the amino acid sequence of SEQ ID NO: 11.

10. The CD48 IgV domain variant of any one of claims 4-7, comprising (a), (b), (c), (d), and (f).

11. The CD48 IgV domain variant of claim 10, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 38.

12. The CD48 IgV domain variant of claim 11, comprising the amino acid sequence of SEQ ID NO: 38.

13. The CD48 IgV domain variant of any one of claims 4-7, comprising (a), (b), (d), and o¬ ld. The CD48 IgV domain variant of claim 13, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 12.

15. The CD48 IgV domain variant of claim 14, comprising the amino acid sequence of SEQ ID NO: 12.

16. The CD48 IgV domain variant of any one of claims 4-7, comprising (a), (d), (e) and (f).

17. The CD48 IgV domain variant of claim 16, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 13.

18. The CD48 IgV domain variant of claim 17, comprising the amino acid sequence of SEQ ID NO: 13.

19. The CD48 IgV domain variant of any one of claims 4-7, comprising (a), (b), (d), (e) and (f).

20. The CD48 IgV domain variant of claim 19, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 14.

21. The CD48 IgV domain variant of claim 20, comprising the amino acid sequence of SEQ ID NO: 14.Attorney Docket No: IMID-102WO22. The CD48 IgV domain variant of any one of claims 4-7, comprising (a), (b), (c), (d), (e) and (f).

23. The CD48 IgV domain variant of claim 22, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 15.

24. The CD48 IgV domain variant of claim 23, comprising the amino acid sequence of SEQ ID NO: 15.

25. The CD48 IgV domain variant of any one of claims 4-7, comprising (a), (c), (d), and (f).

26. The CD48 IgV domain variant of any one of claims 1-25, comprising an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

27. The CD48 IgV domain variant of any one of claims 1-26, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to similarity to SEQ ID NO: 1.

28. The CD48 IgV domain variant of any one of claims 1-27, comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 amino acid mutations relative to SEQ ID NO: 1.

29. The CD48 IgV domain variant of any one of claims 1-28, comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15.

30. The CD48 IgV domain variant of any one of claims 1-29, comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to SEQ ID NO: 15.

31. The CD48 IgV domain variant of any one of claims 1-30, wherein the CD48 IgV domain variant binds CD2 at a higher affinity than a CD48 IgV domain comprising the amino acid sequence of SEQ ID NO: 1.Attorney Docket No: IMID-102WO32. The CD48 IgV domain variant of any one of claims 1-30, wherein the CD48 IgV domain variant binds CD2 at a higher affinity than a corresponding CD48 IgV domain that does not comprise the one or more mutations.

33. The CD48 IgV domain variant of any one of claims 1-32, wherein the CD48 IgV domain variant binds 2B4 at a higher affinity than a CD48 IgV domain comprising the amino acid sequence of SEQ ID NO: 1.

34. The CD48 IgV domain variant of any one of claims 1-32, wherein the CD48 IgV domain variant binds 2B4 at a higher affinity than a corresponding CD48 IgV domain that does not comprise the one or more mutations.

35. A CD48 fragment comprising the CD48 IgV domain variant of any one of claims 1-34.

36. The CD48 fragment of claim 35, further comprising a CD48 IgC2 domain.

37. The CD48 fragment of claim 36, wherein the CD48 IgC2 domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 17.

38. The CD48 fragment of claim 36 or 37, wherein a C-terminus of the CD48 IgV domain is linked, directly or indirectly, to an N-terminus of the CD48 lgC2 domain.

39. The CD48 fragment of claim 38, wherein the C-terminus of the CD48 IgV domain is linked to the N-terminus of the CD48 IgC2 domain via a peptide linker comprising the amino acid sequence of SEQ ID NO: 18.

40. A CD48-Fc fusion polypeptide comprising:(i) the CD48 fragment of any one of claims 35-39; and(ii) an IgG Fc region.

41. The CD48-Fc fusion polypeptide of claim 40, wherein the IgG Fc region is an IgGl, IgG2, IgG3, or IgG4 Fc region.

42. The CD48-Fc fusion polypeptide of claim 41, wherein the IgG Fc region is a human IgGl Fc region.Attorney Docket No: IMID-102WO43. The CD48-Fc fusion polypeptide of claim 42, wherein the human IgGl Fc region comprises one or more mutations that reduce antibody -dependent cellular cytotoxicity (ADCC) effector function.

44. The CD48-Fc fusion polypeptide of claim 42 or 43, wherein the human IgGl Fc region comprises one or more mutations that reduce complement-dependent cytotoxicity (CDC) effector function.

45. The CD48-Fc fusion polypeptide of claim 43 or 44, wherein the human IgGl Fc region comprises one or more mutations at positions 234, 235, 237, 329, 330, and / or 331, according to the EU numbering system.

46. The CD48-Fc fusion polypeptide of claim 45, wherein the human IgGl Fc region comprises one or more mutations selected from L234A, L235A, L235E, G237A, P329A, A330S, and P331S.

47. The CD48-Fc fusion polypeptide of claim 45 or 46, wherein the human IgGl Fc region comprises L234A and L235A substitutions.

48. The CD48-Fc fusion polypeptide of claim 41 , wherein the IgG Fc region is a human IgG4 Fc region.

49. The CD48-Fc fusion polypeptide of any one of claims 40-48, wherein a C-terminus of the CD48 fragment is linked, directly or indirectly, to an N-terminus of the IgG Fc region.

50. The CD48-Fc fusion polypeptide of claim 49, wherein the C-terminus of the CD48 fragment is linked to the N-terminus of the IgG Fc region via a peptide linker.

51. The CD48-Fc fusion polypeptide of claim 50, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 9.

52. A CD48-Fc fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 39.

53. A CD48-Fc fusion polypeptide comprising the amino acid sequence of SEQ ID NO:Attorney Docket No: IMID-102WO54. A CD48-Fc fusion protein comprising two CD48-Fc fusion polypeptides of any one of claims 40-53, wherein the two CD48-Fc fusion polypeptides form a dimer.

55. The CD48-Fc fusion protein of claim 54, wherein the two CD48-Fc fusion polypeptides are linked by one or more covalent bonds.

56. The CD48-Fc fusion protein of claim 55, wherein the one or more covalent bonds are one or more disulfide bonds.

57. The CD48-Fc fusion protein of any one of claims 54-56, wherein the CD48-Fc fusion protein is a soluble protein.

58. A pharmaceutical composition comprising:the CD48 IgV domain variant of any one of claims 1-34, the CD48 fragment of any one of claims 35-39, the CD48-Fc fusion polypeptide of any one of claims 40-53, or the CD48-Fc fusion protein of any one of claims 54-57, anda pharmaceutically acceptable carrier.

59. An isolated nucleic acid encoding the CD48 IgV domain variant of any one of claims 1-34, the CD48 fragment of any one of claims 35-39, or the CD48-Fc fusion polypeptide of any one of claims 40-53.

60. A vector comprising the nucleic acid according to claim 59.

61. A recombinant cell comprising the nucleic acid according to claim 59 or the vector according to claim 60.

62. A method of producing a protein, the method comprising culturing the recombinant cell of claim 61 under conditions to allow expression of the CD48 IgV domain variant, the CD48 fragment, or the CD48-Fc fusion polypeptide encoded by the nucleic acid.

63. The method of claim 62, further comprising purifying the CD48 IgV domain variant, the CD48 fragment, or the CD48-Fc fusion polypeptide.

64. The method of claim 63, further comprising formulating the CD48 IgV domain variant, the CD48 fragment, or the CD48-Fc fusion polypeptide with a pharmaceutically acceptable carrier.Attorney Docket No: IMID-102WO65. A method of inhibiting the activity of a NK cell or a CD8+T cell, the method comprising contacting the NK cell or the CD8+T cell with the CD48 IgV domain variant of any one of claims 1-34, the CD48 fragment of any one of claims 35-39, the CD48-Fc fusion polypeptide of any one of claims 40-53, or the CD48-Fc fusion protein of any one of claims 54-57.

66. A method of treating an autoimmune disease, the method comprising administering to a subject in need thereof an effective amount of the CD48 IgV domain variant of any one of claims 1-34, the CD48 fragment of any one of claims 35-39, the CD48-Fc fusion polypeptide of any one of claims 40-53, the CD48-Fc fusion protein of any one of claims 54-57, or the pharmaceutical composition of claim 58, thereby to treat the autoimmune disease.

67. A method of treating an inflammatory disease, the method comprising administering to a subject in need thereof an effective amount of the CD48 IgV domain variant of any one of claims 1-34, the CD48 fragment of any one of claims 35-39, the CD48-Fc fusion polypeptide of any one of claims 40-53, the CD48-Fc fusion protein of any one of claims 54-57, or the pharmaceutical composition of claim 58, thereby to treat the inflammatory disease.

68. The method of claim 66 or 67, wherein the CD48 IgV domain variant, the CD48 fragment, the CD48-Fc fusion polypeptide, the CD48-Fc fusion protein, or the pharmaceutical composition is administered to the subject intravenously.

69. The method of claim 66 or 67, wherein the CD48 IgV domain variant, the CD48 fragment, the CD48-Fc fusion polypeptide, the CD48-Fc fusion protein, or the pharmaceutical composition is administered to the subject subcutaneously.

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