EA045759B1 - MONOCLONAL ANTIBODIES TO SIRPα AND THEIR APPLICATIONS - Google Patents

Description

State of the art

Signal regulatory protein alpha (SIRPa) is a member of the signal regulatory protein (SIRP) family and also belongs to the immunoglobulin superfamily. Members of the SIRP family are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of receptor tyrosine kinase-related signaling. SIRPa can be phosphorylated by tyrosine kinases. The phosphotyrosine residues of this PTP have been shown to recruit SH2-containing protein tyrosine phosphatases (PTPs) and serve as PTP substrates. SIRPa has been found to be involved in signal transduction mediated by various growth factor receptors. CD47 has been demonstrated to be a ligand. SIRPa has very high similarity to several other members of the SIRP family. SIRPa is expressed primarily by myeloid cells, but also by stem cells or neurons.

SIRPa functions as an inhibitory receptor and interacts with the widely expressed transmembrane protein CD47, also called the don't eat me signal. This interaction negatively regulates the effector function of innate immune cells, such as phagocytosis of host cells. SIRPa diffuses laterally onto the macrophage membrane and accumulates at the phagocytic synapse to bind CD47 and signal its own, which inhibits the cytoskeleton-intensive process of phagocytosis by the macrophage.

Upon CD47 ligation, SIRPa is phosphorylated and recruits phosphatases such as SHP1 and SHP2. The extracellular region contains three domains of the immunoglobulin superfamily - one V-set domain and two C1-set domains of IgSF. SIRPe and γ have similar extracellular structures but different cytoplasmic regions that produce opposing types of signals.

SIRPa recognizes CD47, an antiphagocytic signal that distinguishes living cells from dying cells. The extracellular domain of SIRPa binds to CD47 and transmits intracellular signals through its cytoplasmic domain. CD47 binding is mediated through the NH2-terminal V-like domain of SIRPa.

The cytoplasmic region contains four ITIMs that become phosphorylated upon ligand binding. Phosphorylation mediates the activation of the SHP2 tyrosine kinase. SIRPa also binds the phosphatase SHP1, the adapter protein SCAP2 and the FYN-binding protein. Recruitment of SHP phosphatases to the membrane leads to inhibition of myosin accumulation on the cell surface and provides inhibition of phagocytosis.

Cancer cells express CD47 at high levels, which activates SIRPa and inhibits macrophage-mediated destruction. High-affinity variants of SIRPa that antagonized CD47 on cancer cells were shown to increase phagocytosis of cancer cells. Anti-SIRPa antibodies have also been shown to help macrophages reduce cancer growth and metastasis, alone and synergistically with other cancer treatments.

Brief description of the invention

The present application provides anti-SIRPa antibodies that have high affinity for both v1 and v2 variants, are capable of dose-dependently and effectively blocking the interaction between SIRPa and CD47, and effectively induce macrophage-mediated phagocytosis of CD47-expressing cells. In contrast, known antibodies to SIRPa are capable of recognizing only variant 1.

According to one embodiment, the present invention provides an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein (a) CDRH1 contains the amino acid sequence of SEQ ID NO: 15, CDRH2 contains the amino acid sequence of SEQ ID NO: 16, 21 or 22, CDRH3 contains the amino acid sequence the sequence is SEQ ID NO: 17, CDRL1 contains the amino acid sequence of SEQ ID NO: 18, CDRL2 contains the amino acid sequence of SEQ ID NO: 19 and CDRL3 contains the amino acid sequence of SEQ ID NO: 20; (b) CDRH1 contains the amino acid sequence of SEQ ID NO: 30, CDRH2 contains the amino acid sequence of SEQ ID NO: 31, 36, 37 or 38, CDRH3 contains the amino acid sequence of SEQ ID NO: 32, CDRL1 contains the amino acid sequence of SEQ ID NO: 33, CDRL2 contains the amino acid sequence of SEQ ID NO: 34 and CDRL3 contains the amino acid sequence of SEQ ID NO: 35; (c) CDRH1 contains the amino acid sequence of SEQ ID NO: 47, CDRH2 contains the amino acid sequence of SEQ ID NO: 48, 53 or 54, CDRH3 contains the amino acid sequence of SEQ ID NO: 49, CDRL1 contains the amino acid sequence of SEQ ID NO: 50, CDRL2 contains the amino acid sequence the sequence is SEQ ID NO: 51 and CDRL3 contains the amino acid sequence of SEQ ID NO: 52; (d) CDRH1 contains the amino acid sequence of SEQ ID NO: 63, CDRH2 contains the amino acid sequence of SEQ ID NO: 64, CDRH3 contains amy

- 1045759 the amino acid sequence of SEQ ID NO: 65, CDRL1 contains the amino acid sequence of SEQ ID NO: 66, CDRL2 contains the amino acid sequence of SEQ ID NO: 67 and CDRL3 contains the amino acid sequence of SEQ ID NO: 68; (e) CDRH1 contains the amino acid sequence of SEQ ID NO: 77, CDRH2 contains the amino acid sequence of SEQ ID NO: 78, CDRH3 contains the amino acid sequence of SEQ ID NO: 79, CDRL1 contains the amino acid sequence of SEQ ID NO: 80, CDRL2 contains the amino acid sequence of SEQ ID NO: : 81 and CDRL3 contains the amino acid sequence SEQ ID NO: 82; (f) CDRH1 contains the amino acid sequence of SEQ ID NO: 91, CDRH2 contains the amino acid sequence of SEQ ID NO: 92, CDRH3 contains the amino acid sequence of SEQ ID NO: 93, CDRL1 contains the amino acid sequence of SEQ ID NO: 94, CDRL2 contains the amino acid sequence of SEQ ID NO: : 95 and CDRL3 contains the amino acid sequence SEQ ID NO: 96; or (g) CDRH1 contains the amino acid sequence of SEQ ID NO: 103, CDRH2 contains the amino acid sequence of SEQ ID NO: 104, CDRH3 contains the amino acid sequence of SEQ ID NO: 105, CDRL1 contains the amino acid sequence of SEQ ID NO: 106, CDRL2 contains the amino acid sequence of SEQ ID NO: 107 and CDRL3 contains the amino acid sequence of SEQ ID NO: 108.

In one embodiment, the present invention provides an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 15, CDRH2 contains the amino acid sequence of SEQ ID NO: 16, 21 or 22, CDRH3 contains the amino acid sequence of SEQ ID NO: 17, CDRL1 contains the amino acid sequence of SEQ ID NO: 18, CDRL2 contains the amino acid sequence of SEQ ID NO: 19 and CDRL3 contains the amino acid sequence of SEQ ID NO: 20.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23-27, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: : 1 and 23-27.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-29, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 2 and 28-29.

In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 29.

In yet another embodiment, there is provided an antibody or fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 30, CDRH2 contains the amino acid sequence of SEQ ID NO: 31, 36, 37 or 38, CDRH3 contains the amino acid sequence of SEQ ID NO: 32, CDRL1 contains the amino acid sequence of SEQ ID NO: 33, CDRL2 contains the amino acid sequence of SEQ ID NO: 34 and CDRL3 contains the amino acid sequence of SEQ ID NO: 35.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 39-44, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 3 and 39-44.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 45-46, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 4 and 45-46.

In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 43, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 45.

In yet another embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region containing the defining com

- 2 045759 tribalism of the heavy chain regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 47, CDRH2 contains the amino acid sequence of SEQ ID NO: : 48, 53 or 54, CDRH3 contains the amino acid sequence of SEQ ID NO: 49, CDRL1 contains the amino acid sequence of SEQ ID NO: 50, CDRL2 contains the amino acid sequence of SEQ ID NO: 51 and CDRL3 contains the amino acid sequence of SEQ ID NO: 52.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 55-60, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: : 5 and 55-60.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 61-62, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 6 and 61-62.

In some embodiments, an antibody or fragment disclosed herein is capable of binding to SIRPa variant 1 and variant 2. In some embodiments, the antibody or fragment is humanized. In some embodiments, an antibody or fragment disclosed herein additionally has binding specificity for a second target protein.

Also provided in some embodiments are compositions comprising an antibody or fragment thereof and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a second antibody having specificity for a tumor antigen. In some embodiments, the second antibody is a tumor opsonizing antibody.

Methods and uses for treating diseases and conditions are also provided. In one embodiment, there is provided a method of treating cancer in a patient in need thereof, comprising administering to said patient an antibody or fragment thereof of the present invention.

Brief description of graphic materials

In fig. Figure 1 shows cross-linking of SIRPa v1 and v2 antibodies.

In fig. 2 shows the binding affinity of the antibody to SIRPa v1.

In fig. Figure 3 shows the competition of antibodies with CD47 for interaction with SIRPa.

In fig. Figure 4 shows antibody induction of macrophage-mediated phagocytosis.

In fig. Figure 5 shows an increase in macrophage-mediated phagocytosis of tumor cells due to antibody treatment.

In fig. Figure 6 shows animal renderings and diagrams demonstrating that 03-hz51 interacted synergistically with rituximab to cause complete inhibition of tumor growth in a model of Raji lymphoma.

Detailed Description of the Invention

Definitions

It should be noted that a term in the singular refers to one or more objects expressed by such a term; for example, by antibody is meant one or more antibodies. Accordingly, the terms singular, one or more, and at least one may be used interchangeably herein.

As used herein, the term polypeptide is intended to include the singular polypeptide as well as a plurality of polypeptides, and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of product. Thus, peptides, dipeptides, tripeptides, oligopeptides, protein, amino acid chain, or any other term used to designate a chain or chains of two or more amino acids are covered by the definition of polypeptide, and the term polypeptide may be used in place of or interchangeably with any of these terms . The term polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolysis, or modification with non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a given nucleic acid sequence. It can be obtained by any method, including chemical synthesis.

Homology or identity or similarity refers to the sequence similarity of two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. When

- 3 045759 the position in the compared sequence is occupied by the same base or amino acid, the molecules in this position are homologous. The degree of sequence homology depends on the number of matching or homologous positions shared by the sequences. An unrelated or non-homologous sequence is less than 40% identical, preferably less than 25%, identical to one of the sequences according to the present invention.

If a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of sequence identity with another sequence, this means that in a sequence alignment, that percentage of bases (or amino acids) when comparing the two sequences is the same.

The term equivalent nucleic acid or polynucleotide refers to a nucleic acid having a nucleotide sequence with some degree of homology or identity to the nucleotide sequence of the nucleic acid or its complement. A double-stranded nucleic acid homolog is intended to include nucleic acids with a nucleotide sequence that is somewhat homologous to it or its complement. In one aspect, nucleic acid homologues are capable of hybridizing with a nucleic acid or its complement. Likewise, an equivalent polypeptide refers to a polypeptide with some degree of homology or amino acid sequence identity to the reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide contains one, two, three, four, or five insertions, deletions, substitutions, and combinations thereof compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (eg, epitope binding) or structure (eg, salt bridge) of the reference sequence.

As used herein, an antibody or antigen-binding polypeptide refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody can be the whole antibody and any antigen binding fragment or one chain thereof. Thus, the term antibody includes any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule having antigen binding biological activity. Examples thereof include, but are not limited to, a heavy or light chain complementarity determining region (CDR) or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one part of the binding protein.

The terms antibody fragment or antigen binding fragment as used herein are a portion of an antibody such as F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv and the like. Regardless of structure, the antibody fragment binds to the same antigen that is recognized by the intact antibody. The term antibody fragment includes aptamers, spiegelmers and diabodies. The term antibody fragment also includes any synthetic or genetically modified protein that acts as an antibody by binding to a specific antigen to form a complex.

Single chain variable fragment or scFv refers to a fusion protein of the variable regions of the heavy (VH) and light ( _VL ) chains of immunoglobulins. In some aspects, the regions are connected by a short linker peptide ranging from 10 to about 25 amino acids in length. The linker may be high in glycine for flexibility and serine or threonine for solubility, and can either connect the N-terminus of VH to the C-terminus of _VL or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of constant regions and the introduction of a linker. scFv molecules are known in the art and are described, for example, in US Pat. No. 5,892,019.

The term antibody covers various broad classes of polypeptides that are biochemically distinct. Those skilled in the art will recognize that heavy chains are classified into gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), of which there are also several subclasses (eg, γ1-γ4). It is the nature of this chain that determines the class of the antibody as IgG, IgM, IgA IgG or IgE, respectively. Subclasses (isotypes) of immunoglobulins, such as IgG1, IgG2, _IgG3 , IgG4, _IgG5 , etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible by one skilled in the art given the present disclosure and are accordingly within the scope of the present invention. All classes of immunoglobulins are clearly within the scope of the present invention, and the following discussion will largely concern the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 Da and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are typically linked by disulfide bonds in a Y configuration, where the light chains enclose the heavy chains starting

- 4 045759 at the mouth of Y and continuing through the variable region.

Antibodies, antigen-binding polypeptides, variants or derivatives thereof of the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments, for example, Fab, Fab' and F( ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), VK or VH domain-containing fragments, Fab expression library-derived fragments, and anti-idiotypic (anti- Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules of the present invention can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of the immunoglobulin molecule.

Light chains are classified as kappa or lambda (K, λ). Each class of heavy chain can be associated with a kappa or lambda light chain. Typically, the light and heavy chains are covalently linked to each other, and the tail portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when immunoglobulin molecules are produced by hybridomas, B cells or genetically modified host cells. In the heavy chain, the amino acid sequences run from the N-terminus at the bifurcated ends in a Y-shaped configuration to the C-terminus at the bottom of each chain.

Both light and heavy chains are divided into regions of structural and functional homology. The terms constant and variable are used in relation to a function. In this regard, it should be understood that the variable domains of both the light (VK) and heavy (VH) chain portions determine antigen recognition and specificity. In contrast, the light chain (CK) and heavy chain constant domains (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental transport, Fc receptor binding, complement fixation and the like. It is accepted that the numbers of constant region domains increase with distance from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxy terminus of the heavy and light chains, respectively.

As stated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK domain and the VH domain, or a subset of complementarity determining regions (CDRs), of an antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen binding site present at the end of each Y arm. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VK chains (i.e. CDR-H1, CDR-H2, CDRH3, CDR-L1 , CDR-L2 and CDR-L3). In some cases, such as certain immunoglobulin molecules derived from camelids or genetically engineered from camelid immunoglobulins, the complete immunoglobulin molecule may consist of only heavy chains and no light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

In naturally occurring antibodies, the six complementarity determining regions, or CDRs, present in each antigen binding domain are short, non-contiguous sequences of amino acids arranged in a specific manner to form the antigen binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domains, called framework regions, exhibit less intermolecular variability. The framework regions generally adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases are part of, the β-sheet structure. Thus, the framework regions form a framework that ensures that the CDRs are positioned in the correct orientation through interchain non-covalent interactions. The antigen-binding domain, formed by the CDRs that occupy their positions, defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to the recognized epitope. The amino acids constituting the CDRs and framework regions, respectively, can be easily determined for any given heavy or light chain variable region by one skilled in the art since they have been precisely determined (see Sequences of Proteins of Immunological Interest, Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917(1987)).

In the event that there are two or more definitions of a term that is used and/or accepted in the art, the definition of the term used in this application is intended to include all such meanings unless expressly stated otherwise. A specific example is the use of the term complementarity determining region (CDR) to describe non-contiguous antigen binding sites present in the variable region of both heavy and light chain polypeptides. This particular site has been described by Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196:901-917

- 5 045759 (1987), incorporated herein by reference in its entirety. The Kabat and Chothia definitions of CDRs include overlapping or subsets of amino acid residues when compared to each other. However, the use of any of the terms in the context of a CDR antibody or variants thereof is intended to be within the scope of that term as defined and used herein. A comparison of the corresponding amino acid residues comprising the CDRs as defined in each of the above references is shown in the table below. The exact residue numbers comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues form a particular CDR given the known amino acid sequence of the antibody variable region.

Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One skilled in the art can unambiguously apply this Kabat numbering system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, the term Kabat numbering system refers to the numbering system formulated by Kabat et al., U.S. Dept. of Health and Human Services, Sequence of Proteins of Immunological Interest (1983).

In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately 31 amino acids (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next residue tryptophan. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, contains approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins approximately at the thirty-third amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends with the sequence W-G-X-G, where X represents any amino acid. CDR-L1 starts at approximately residue 24 (ie, after the cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty-third residue after the end of CDR-L2 (ie, after the cysteine residue); contains approximately 7-11 residues and ends with the sequence F or W-G-X-G, where X is any amino acid.

The antibodies disclosed herein can be derived from any animal, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In yet another embodiment, the variable region may be derived from cartilaginous fish (eg, sharks).

As used herein, the term heavy chain constant region includes amino acid sequences derived from the immunoglobulin heavy chain. The heavy chain constant region-containing polypeptide contains at least one of the following: a CH1 domain, a hinge (eg, upper, middle, and/or lower hinge) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use in the invention may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain containing a CH1 domain, at least part of a hinge domain and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In yet another embodiment, the polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. In addition, an antibody for use in the invention may lack at least a portion of the CH2 domain (eg, all or part of the CH2 domain). As stated above, one skilled in the art will appreciate that the heavy chain constant region can be modified such that its amino acid sequence differs from the naturally occurring immunoglobulin molecule.

The heavy chain constant region of the antibody disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG ₃ molecule. In yet another example, the heavy chain constant region may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG ₃ molecule. In yet another example, the heavy chain portion may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG ₄ molecule.

As used herein, the term light chain constant region includes amino acid sequences derived from the light chain of an antibody. Preferably, the light chain constant region comprises at least one of a kappa constant domain or a lambda constant domain.

A light-heavy chain pair refers to a combination of a light chain and a heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

As noted earlier, the subunit structures and three-dimensional configuration of the constant regions of the various classes of immunoglobulins are well known. As used herein, the term VH domain includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term CH1 domain includes the first (most amino-terminal) domain of the immunoglobulin heavy chain constant region. The CH1 domain is adjacent to the VH domain and is amino-terminal relative to the hinge region of the immunoglobulin heavy chain molecule.

As used herein, the term CH2 domain includes the portion of the heavy chain molecule that extends, for example, from approximately residue 244 to residue 360 of an antibody, as indicated by conventional numbering schemes (residues 244-360, Kabat numbering system, and residues 231-340 , EU numbering system, see Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983)). The CH2 domain is unique in that it is not closely associated with another domain. Rather, two N-linked branched carbohydrate chains are located between the two CH2 domains of the intact native IgG molecule. It is also well described that the CH3 domain extends from the CH2 domain to the end of the IgG molecule and contains approximately 108 residues.

As used herein, the term hinge region includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently of each other. The hinge regions can be divided into three distinct domains: the upper, middle and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

As used herein, the term disulfide bond includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term chimeric antibody refers to any antibody in which the immunoreactive region or site is derived from or derived from a first species and the constant region (which may be intact, partial, or modified in accordance with the present invention) is derived from a second species. In certain embodiments, the target binding region or site will be derived from a non-human source (eg, mouse or primate) and the constant region will be human.

As used herein, the percentage of humanization is calculated by determining the amount of backbone amino acid difference (i.e., difference in regions other than the CDR) between the humanized domain and the germline domain, subtracting that amount from the total number of amino acids, and then dividing that difference by total number of amino acids and multiply by 100.

By specifically binds or has specificity for, it is generally meant that the antibody binds to an epitope via its antigen binding domain, and that binding implies some complementarity between the antigen binding domain and the epitope. By this definition, an antibody is said to specifically bind to an epitope when it binds to that epitope through its antigen binding domain more likely than it would to a random, unrelated epitope. The term specificity is used herein to assess the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody A may be said to have a higher specificity for a given epitope than antibody B, or antibody A may be said to bind epitope C with a higher specificity than its specificity for the related epitope D.

As used herein, the terms treat or treatment refer to both therapeutic treatment and prophylactic or prophylactic measures where the goal is to prevent or slow (reduce) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desirable clinical outcomes include, but are not limited to, partial relief of symptoms, reduction in disease severity, stable (i.e., not worsening) disease, delay or slowing of disease progression, reduction or temporary relief of disease, and remission (partial or complete). ), both detectable and undetectable. Treatment may also mean longer survival than would be expected if no treatment was given. Subjects in need of treatment include those already having the condition or disorder, as well as those who are predisposed to developing the condition or disorder, or those whose condition or disorder needs to be prevented.

By subject or individual or animal or patient or mammal is meant any subject, particularly a mammal, for which diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, domesticated animals, farm and zoo animals, sports animals, or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like. Further.

As used herein, phrases such as a patient in need of treatment or a subject in need of treatment include subjects, such as mammalian subjects, who would benefit from administration of an antibody or composition of the present invention in use for, for example, a detection, diagnostic procedure and/or for treatment.

Antibodies to SIRPa

The present invention provides anti-SIRPa antibodies and fragments that have high affinity for both v1 and v2 variants. Variant 1 (hSIRPaV1) is the dominant variant among Europeans, Africans, mixed-race Americans, and South Asians. Variant 2 (hSIRPaV2) is the dominant variant among East Asians. The sequences of hSIRPaV1 and hSIRPaV2 differ within the extracellular Ig-like V-like (IgV) domain. The ability of the antibodies and fragments disclosed herein to recognize both variants makes them effective in the broadest patient population.

In addition, the antibodies and fragments disclosed herein demonstrate excellent binding affinity, potent induction of macrophage-mediated phagocytosis, and manufacturing suitability in terms of superior chemical properties, manufacturing process, and quality control (CMC).

Thus, according to one embodiment of the present invention, antibodies and antigen-binding fragments thereof are provided that are capable of binding to both SIRPa variants 1 and 2. Examples of antibodies include the murine antibodies listed in table. 1, as well as humanized antibodies from table. 2-8.

Also included are antibodies that contain the same CDRs as illustrated herein. In some embodiments, the disclosed antibodies and fragments include antibodies and fragments that bind to the same epitope as those illustrated herein, and antibodies and fragments that compete with the disclosed antibodies and fragments for binding to SIRPa.

According to one embodiment, the present invention provides an antibody or fragment thereof comprising heavy chain and light chain variable domains with CDR regions disclosed herein, as well as biological equivalents thereof.

In one embodiment, the CDRs are CDRs 248G3F6, as shown in Tables 2B and 2D. In one embodiment, CDRH1 contains the amino acid sequence of SEQ ID NO: 15 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH2 contains the amino acid sequence of SEQ ID NO: 16, 21 or 22 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH3 contains the amino acid sequence SEQ ID NO: 17 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL1 contains the amino acid sequence SEQ ID NO: 18 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL2 contains the amino acid sequence of SEQ ID NO: 19 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, and CDRL3 contains the amino acid sequence of SEQ ID NO: 20 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof.

In one embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a variable a light chain region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 15, CDRH2 contains the amino acid sequence of SEQ ID NO: 16, CDRH3 contains the amino acid sequence of SEQ ID NO: 17, CDRL1 contains the amino acid sequence of SEQ ID NO: 18, CDRL2 contains the amino acid sequence of SEQ ID NO: 19 and CDRL3 contains the amino acid sequence of SEQ ID NO: 20.

In one embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a variable a light chain region containing the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 15, CDRH2 contains the amino acid sequence of SEQ ID NO: 21, CDRH3 contains the amino acid sequence of SEQ ID NO: 21 17, CDRL1 contains the amino acid sequence of SEQ ID NO: 18, CDRL2 contains the amino acid sequence of SEQ ID NO: 19, and CDRL3 contains the amino acid sequence of SEQ ID NO: 20.

In one embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a variable a light chain region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 15, CDRH2 contains the amino acid sequence of SEQ ID NO: 22, CDRH3 contains the amino acid sequence of SEQ ID NO: 17, CDRL1 contains the amino acid sequence of SEQ ID NO: 18, CDRL2 contains the amino acid sequence of SEQ ID NO: 19 and CDRL3 contains the amino acid sequence of SEQ ID NO: 20.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23-27, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: : 1 and 23-27.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-29, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 2 and 28-29.

In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 29.

In one embodiment, the CDRs are CDR 300A6A6, as shown in Tables 3B and 3D. In one embodiment, CDRH1 contains the amino acid sequence of SEQ ID NO: 30 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH2 contains the amino acid sequence of SEQ ID NO: 31, 36, 37 or 38 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH3 contains the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL1 contains the amino acid sequence of SEQ ID NO: 33 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL2 contains the amino acid sequence of SEQ ID NO: 34 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, and CDRL3 contains the amino acid sequence of SEQ ID NO: 35 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof.

In one embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a variable a light chain region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 30, CDRH2 contains the amino acid sequence of SEQ ID NO: 31, CDRH3 contains the amino acid sequence of SEQ ID NO: 32, CDRL1 contains the amino acid sequence of SEQ ID NO: 33, CDRL2 contains the amino acid sequence of SEQ ID NO: 34 and CDRL3 contains the amino acid sequence of SEQ ID NO: 35.

In one embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a variable a light chain region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 30, CDRH2 contains the amino acid sequence of SEQ ID NO: 36, CDRH3 contains the amino acid sequence of SEQ ID NO: 32, CDRL1 contains the amino acid sequence of SEQ ID NO: 33, CDRL2 contains the amino acid sequence of SEQ ID NO: 34 and CDRL3 contains the amino acid sequence of SEQ ID NO: 35.

In one embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody

- 9 045759 or a fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 30, CDRH2 contains the amino acid sequence of SEQ ID NO: 37, CDRH3 contains the amino acid sequence of SEQ ID NO: 32, CDRL1 contains the amino acid sequence of SEQ ID NO: 33, CDRL2 contains the amino acid sequence of SEQ ID NO: 34 and CDRL3 contains the amino acid sequence of SEQ ID NO: 35.

In one embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a variable a light chain region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 30, CDRH2 contains the amino acid sequence of SEQ ID NO: 38, CDRH3 contains the amino acid sequence of SEQ ID NO: 32, CDRL1 contains the amino acid sequence of SEQ ID NO: 33, CDRL2 contains the amino acid sequence of SEQ ID NO: 34 and CDRL3 contains the amino acid sequence of SEQ ID NO: 35.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 39-44, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 3 and 39-44.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 45-46, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 4 and 45-46.

In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 43 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 45.

In one embodiment, the CDRs are CDR 102A10F2, as shown in Table. 4B and 4D. In one embodiment, CDRH1 contains the amino acid sequence of SEQ ID NO: 47 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH2 contains the amino acid sequence of SEQ ID NO: 48, 53 or 54 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH3 contains the amino acid sequence of SEQ ID NO: 49 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL1 contains the amino acid sequence of SEQ ID NO: 50 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL2 contains the amino acid sequence of SEQ ID NO: 51 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, and CDRL3 contains the amino acid sequence of SEQ ID NO: 52 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof.

In one embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a variable a light chain region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 47, CDRH2 contains the amino acid sequence of SEQ ID NO: 48, CDRH3 contains the amino acid sequence of SEQ ID NO: 49, CDRL1 contains the amino acid sequence of SEQ ID NO: 50, CDRL2 contains the amino acid sequence of SEQ ID NO: 51 and CDRL3 contains the amino acid sequence of SEQ ID NO: 52.

In one embodiment, an antibody or fragment thereof is provided having specificity for binding to wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a variable a light chain region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 47, CDRH2 contains the amino acid sequence of SEQ ID NO: 53, CDRH3 contains the amino acid sequence of SEQ ID NO: 49, CDRL1 contains the amino acid sequence of SEQ ID NO: 50, CDRL2 contains the amino acid sequence of SEQ ID NO: 51 and CDRL3 contains the amino acid sequence of SEQ ID NO: 52.

- 10 045759

In one embodiment, an antibody or fragment thereof is provided having binding specificity for wild-type human signal regulatory protein alpha (SIRPa), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a variable a light chain region comprising the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and wherein CDRH1 contains the amino acid sequence of SEQ ID NO: 47, CDRH2 contains the amino acid sequence of SEQ ID NO: 54, CDRH3 contains the amino acid sequence of SEQ ID NO: 49, CDRL1 contains the amino acid sequence of SEQ ID NO: 50, CDRL2 contains the amino acid sequence of SEQ ID NO: 51 and CDRL3 contains the amino acid sequence of SEQ ID NO: 52.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 55-60, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: : 5 and 55-60.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 61-62, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 6 and 61-62.

In one embodiment, the CDRs are CDR 62D2H6, as shown in Tables 5B and 5D. In one embodiment, CDRH1 contains the amino acid sequence of SEQ ID NO: 63 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH2 contains the amino acid sequence of SEQ ID NO: 64 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH3 contains the amino acid sequence SEQ ID NO: 65 or a variant thereof, containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL1 contains the amino acid sequence SEQ ID NO: 66 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL2 contains the amino acid sequence of SEQ ID NO: 67 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, and CDRL3 contains the amino acid sequence of SEQ ID NO: 68 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 69-72, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 7 and 69-72.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 73-76, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 8 and 73-76.

In one embodiment, the CDRs are CDR 211F8E11, as shown in Tables 6B and 6D. In one embodiment, CDRH1 contains the amino acid sequence of SEQ ID NO: 77 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH2 contains the amino acid sequence of SEQ ID NO: 78 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH3 contains the amino acid sequence SEQ ID NO: 79 or a variant thereof, containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL1 contains the amino acid sequence SEQ ID NO: 80 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL2 contains the amino acid sequence of SEQ ID NO: 81 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, and CDRL3 contains the amino acid sequence of SEQ ID NO: 82 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 83-86, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: : 9 and 83-86.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 87-90, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 10 and 87-90.

In one embodiment, the CDRs are CDR 217D11E5, as shown in Tables 7B and 7D. In one embodiment, CDRH1 contains the amino acid sequence of SEQ ID NO: 91 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH2 contains the amino acid sequence of SEQ ID NO: 92 or a variant thereof containing one, two or three deletions, insertions, substitutions, or combinations thereof, CDRH3 contains the amino acid sequence of SEQ ID NO: 93 or a variant thereof containing one, two or three deletions, insertions, substitutions

- 11 045759 or combinations thereof, CDRL1 contains the amino acid sequence SEQ ID NO: 94 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL2 contains the amino acid sequence SEQ ID NO: 95 or a variant thereof containing one , two or three deletions, insertions, substitutions, or combinations thereof, and CDRL3 contains the amino acid sequence of SEQ

ID NO: 96 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 97-100, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 11 and 97-100.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 101-102, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 101-102.

In one embodiment, the CDRs are CDR 234B7D5, as shown in Tables 8B and 8D. In one embodiment, CDRH1 contains the amino acid sequence of SEQ ID NO: 103 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH2 contains the amino acid sequence of SEQ ID NO: 104 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRH3 contains the amino acid sequence SEQ ID NO: 105 or a variant thereof, containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL1 contains the amino acid sequence SEQ ID NO: 106 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, CDRL2 contains the amino acid sequence of SEQ ID NO: 107 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof, and CDRL3 contains the amino acid sequence of SEQ ID NO: 108 or a variant thereof containing one, two or three deletions, insertions, substitutions or combinations thereof.

In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 109-112, or a peptide that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs : 13 and 109-112.

In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 113-118, or a peptide at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: : 14 and 113-118.

Antibodies that contained these CDR regions, whether murine, humanized, or chimeric, had potent SIRPa binding and inhibition activity. As shown in Example 5, certain residues in the CDR can be modified to maintain or improve properties or reduce the likelihood of post-translational modifications (PTMs). Such modified CDRs may be referred to as affinity matured CDRs or risk reduced CDRs.

Non-limiting examples of reduced risk CDRs are presented in Table. 2B, 3B and 4B. Modified CDRs may include CDRs that have an insertion, deletion, and/or substitution of one, two, or three amino acids. In some embodiments, the substitutions may be conservative substitutions.

A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. The art has identified families of amino acid residues containing similar side chains, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid chain may be replaced by a structurally similar chain that differs in the order and/or composition of the side chain family members.

Non-limiting examples of conservative amino acid substitutions are presented in the table below, where a similarity score of 0 or higher indicates a conservative substitution between two amino acids.

- 12 045759

Table A. Amino acid similarity matrix

With G R S A T D E N Q N TO R V M | L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 1 -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 TO -5 -2 -1 0 -1 0 0 0 1 1 0 5 N -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 R -3 -1 6 G -3 5 WITH 12

Table B. Conservative amino acid substitutions

For amino acid Change to Alanin D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gin, D-GIn Aspartic acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gin, D-GIn Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser Glutamine D-GIn, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-GIn Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-Ala Isoleucine D-lle, Vai, D-Val, Leu, D-Leu, Met, D-Met Leucine Vai, D-Val, Met, D-Met, D-lle, D-Leu, lie Lysine D-Lys, Arg, D-Arg, Orn, D-Orn Methionine D-Met, S-Me-Cys, lie, D-lle, Leu, D-Leu, Vai, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-Pro Serin D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Vai, D-Val Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valin D-Val, Leu, D-Leu, lie, D-lle, Met, D-Met

One skilled in the art will also appreciate that the antibodies disclosed herein may be modified such that their amino acid sequence differs from the naturally occurring binding polypeptide from which they are derived. For example, the polypeptide or amino acid sequence derived from the designated protein may be similar, e.g. have a certain percentage identity to the original sequence, e.g. it may be 60%, 70%, 75%, 80%, 85%, 90%, 95% , 98% or 99% identical to the original sequence.

In certain embodiments, the antibody contains an amino acid sequence or one or more fragments that are not typically associated with the antibody. Examples of modifications are described in more detail below. For example, an antibody of the invention may contain a flexible linker sequence or may be modified to add a functional moiety (eg, PEG, drug, toxin, or label).

Antibodies, variants or derivatives thereof according to the invention include derivatives that have been modified, i.e., by covalently attaching a molecule of any type to the antibody in such a way that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not limited to, antibodies can be modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolysis, binding to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be accomplished by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, antibodies may contain one or more non-canonical amino acids.

In some embodiments, antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEGs.

Antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radiolabels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin. , an ultrasound image contrast enhancing agent, a non-radioactive tracer, a combination thereof, and other similar agents known in the art.

Antibodies can be labeled for detection by binding them to a chemiluminescent compound. The presence of a chemiluminescent compound-labeled antigen-binding polypeptide is then determined by detecting the luminescence produced by the chemical reaction. Examples of compounds particularly suitable as a chemiluminescent label are luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Antibodies can also be labeled for detection using metals that emit fluorescent radiation, such as ¹⁵² Eu or other metals of the lanthanide family. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known, see, for example, Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., Antibodies For Drug Delivery, in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303- 16 (1985), and Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, Immunol. Rev. (52:119-58 (1982)).

Bifunctional molecules and combination therapies

Macrophages are an important component of the innate immune system. Macrophages inhibit tumor growth through phagocytosis of tumor cells. Tumor cells can evade macrophage surveillance by activating the don't eat me signal - CD47 - on their cell surface. CD47 is expressed ubiquitously but is up-regulated in various tumor types. By interacting with various ligands, CD47 plays a role in the regulation of cell motility, adhesion, migration and platelet activation. SIRPa is another member of the immunoglobulin superfamily and is primarily expressed on the surface of neurons and myeloid cells, including macrophages, granulocytes, monocytes and dendritic cells. The CD47/SIRPa axis is a known major pathway for tumor cells to evade immune surveillance.

Agents targeting CD47 can initiate antibody opsonization and activate antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), which promotes tumor cell destruction. However, due to its ubiquitous expression, CD47 can lead to erythrotoxicity and other hematological side effects due to the presence of the antigen on blood cells. Molecules targeting SIRPa, or bispecific agents targeting both SIRPa and CD47, can be designed to induce the effect of ADCC and ADCP through the presence of functional Fc domains.

Because SIRPa is more restricted in expression compared to CD47, these molecules should not have as many hematological complications compared to molecules targeting CD47 alone. Thus, anti-SIRPa antibodies may be safer than anti-CD47 antibodies. Another option is the development of bispecific agents targeting both the CD47/SIRPa axis and another tumor antigen. These agents may be bispecific antibodies, fusion proteins, or combination therapies.

Examples of tumor antigens, particularly those that can cause tumor opsonization, include, but are not limited to, CD19, CD20, EGFR, HER2, CD3, CD16, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR , CD73, CD39, CD40, CEA, HER2, VEGFR, TIGIT, claudin 18.2, CD24, GPC3, Il13RA2, 4-1BB, CCR8 and CMET.

Another format of bispecific antibodies has also been proposed. In some embodiments, the SIRPa fragment and the second fragment are each independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc moiety.

Antibody-encoding polynucleotides and methods for producing antibodies

The present invention also relates to isolated polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof according to the invention. The polynucleotides of the present invention may encode entire variable regions of the heavy and light chains of antigen binding polypeptides, variants or derivatives thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of the present invention may encode portions of the heavy and light chain variable regions of antigen binding polypeptides, variants or derivatives thereof, on the same polynucleotide molecule or on separate polynucleotide molecules.

Methods for producing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen binding polypeptides of the present invention are entirely human. Fully human antibodies can be obtained using techniques described in the art and in this application. For example, fully human antibodies against a particular antigen can be obtained by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen stimulation while its endogenous loci have been inactivated. Examples of techniques that can be used to produce such antibodies are described in US Pat. Nos. 6,150,584, 6,458,592, 6,420,140, incorporated by reference in their entirety.

Treatment methods

As described herein, the antibodies, variants or derivatives of the present invention can be used in certain methods of treatment and diagnosis.

The present invention further relates to methods of antibody therapy comprising administering antibodies of the invention to a patient, such as an animal, mammal and human, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the invention (including variants and derivatives thereof as described herein).

The antibodies of the invention may also be used to treat or inhibit cancer. As stated above, SIRPa can be overexpressed in tumor cells, particularly in gastric, pancreatic, esophageal, ovarian and lung tumors. Inhibition of SIRPa has been shown to be effective in the treatment of tumors.

Accordingly, some embodiments provide methods for treating cancer in a patient in need thereof. The method in one embodiment includes administering to said patient an effective amount of an antibody of the present invention. In some embodiments, at least one of the cancer cells (eg, stromal cells) in the patient overexpresses SIRPa.

Cell therapy methods, such as chimeric antigen receptor (CAR) T cell therapy, are also covered by the present invention. A suitable cell that is contacted with an anti-SIRPa antibody of the present invention (or, alternatively, genetically modified to express an anti-SIRPa antibody of the present invention) may be used. After such contact or genetic modification, the cell can then be administered to a cancer patient in need of treatment. The cancer patient may have any type of cancer described herein. The cell (eg, a T cell) may be, for example, a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or a combination thereof, but is not limited to.

In some embodiments, the cell was isolated from the cancer patient himself. In some embodiments, the cell was provided by a donor or from a cell bank. When a cell is isolated from a cancer patient, unwanted immune reactions can be minimized.

Non-limiting examples of cancers include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer and thyroid cancer. In some embodiments, the cancer is one or more of gastric, pancreatic, esophageal, ovarian, and lung cancer.

Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed, and/or predicted by antibodies or variants or derivatives thereof of the invention include, but are not limited to, cancer progression and/or metastasis and associated disorders such as leukemia (including acute leukemia (eg, acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia, and erythroleukemia)) and chronic leukemia (eg, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia goats) , polycythemia vera, lymphomas (eg, Hodgkin's disease and non-Hodgkin's lymphoma), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease and solid tumors, including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic

- 15 045759 sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma , adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer uterine cancer, testicular tumor , lung carcinoma, small cell carcinoma of the lung, uric bubble carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharymia, ependema, pinealoma, hemangiwhistoma, acoustic neurinoma, oligodendrolyme, meningioma, melanoma, neo -nominal and nerm Tinoblastoma.

The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the specific antibody, variant or derivative used, age, body weight, general health, gender and diet of the patient, as well as the time of administration, rate of elimination, combination of prescribed medications and the severity of the specific disease being treated. The assessment of such factors by medical professionals is within the normal skill of one skilled in the art. The amount will also depend on the particular patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease and the desired effect. The amount used can be determined using pharmacological and pharmacokinetic principles well known in the art.

Routes of administration of antibodies or variants thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding polypeptides or compositions can be administered by any suitable route, for example, by infusion or bolus injection, by absorption through the epithelial lining or mucosa (eg, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (for example, by powders, ointments, drops or transdermal patch), buccally, or by oral or nasal administration. spray.

As used herein, the term parenteral refers to routes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.

Administration may be systemic or local. In addition, it may be desirable to administer the antibodies of the invention to the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be simplified by using an intraventricular catheter, for example, attached to a reservoir such as an Ommaya reservoir. Pulmonary administration may also be used, for example, using an inhaler or nebulizer and a nebulizing agent formulation.

It may be desirable to administer the antigen-binding polypeptides or compositions of the invention locally to the area in need of treatment; this may be achieved, for example, but not limited to, by local infusion during surgery, topical application, for example in combination with wound dressing after surgery, by injection, by catheter, by suppository or by implant, wherein said the implant may be made of porous, non-porous or gel-like material, including membranes such as Sikalastic membranes, or fibers. Preferably, when administering a protein, including an antibody, according to the invention, care must be taken to ensure that non-protein absorbent materials are used.

The amount of antibodies of the invention that will be effective for treating, inhibiting and preventing an inflammatory, immune or malignant disease, disorder or condition can be determined by standard clinical methods. Additionally, in vitro assays may optionally be used as aids in establishing optimal dosage ranges. The exact dosage to be used in a drug will also depend on the route of administration and the severity of the disease, disorder or condition, and should be selected in accordance with the judgment of the practitioner and the specifics of each patient. Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

As a general guideline, the dosage of the antigen binding polypeptides of the present invention administered to a patient is typically 0.1 to 100 mg/kg patient body weight, 0.1 to 20 mg/kg patient body weight, or 1 to 10 mg/kg patient's body weight. In general, human antibodies have a longer half-life in the human body than antibodies originating

- 16 045759 from other species, which is associated with an immune response to foreign polypeptides. Thus, it is often possible to use lower dosages of human antibodies and lower frequency of administration.

In addition, the dosage and frequency of administration of antibodies according to the invention can be reduced by enhancing the uptake and penetration of antibodies into tissues (eg, brain) through modifications such as, for example, lipidation.

In a further embodiment, the compositions of the invention are administered in combination with cytokines. Cytokines that can be administered with the compositions of the invention include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL -13, IL-15, anti-CD40, CD40L and TNF-a.

In additional embodiments, the compositions of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

Diagnostic methods

Overexpression of SIRPa is observed in some tumor samples, and patients having SIRPa overexpressing cells are likely to respond to treatment with anti-SIRPa antibodies according to the present invention. Accordingly, the antibodies of the present invention can also be used for diagnostic and prognostic purposes.

The sample, which preferably includes a cell, may be obtained from a patient, which may be a cancer patient or a patient awaiting diagnosis. The cell may be a cell from a tumor tissue or tumor block, a blood sample, a urine sample, or any sample from a patient. After optional pre-treatment of the sample, it can be incubated with the antibody of the present invention under conditions that allow the antibody to interact with the SIRPa protein potentially present in the sample. Methods such as ELISA, which use an anti-SIRPa antibody to detect the presence of SIRPa protein in a sample, can be used.

Determining the presence of SIRPa protein in a sample (optionally with quantitation or concentration) can be used to diagnose cancer, as evidence that a patient is eligible for antibody treatment, or as evidence that a patient has responded (or not responded) to cancer treatment. For a predictive method, detection may be performed one, two or more times, at certain stages, after the start of cancer treatment, to check the progress of treatment.

Compositions

The present invention also relates to pharmaceutical compositions. Such compositions contain an effective amount of the antibody and an acceptable carrier. In some embodiments, the composition further includes a second anticancer agent (eg, an immune checkpoint inhibitor).

In a particular embodiment, the term pharmaceutically acceptable means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia as suitable for use in animals and, more particularly, in humans. In addition, the pharmaceutically acceptable carrier is typically a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of adjuvant composition.

The term carrier refers to a diluent, adjuvant, excipient or excipient with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier for intravenous administration of a pharmaceutical composition. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol and the like. The composition may optionally also contain minor amounts of wetting or emulsifying agents or buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methylparabens are also provided; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity, such as sodium chloride or dextrose. These compositions may take the form of solutions, suspensions, emulsions, tablets, dragees, capsules, powders, sustained release formulations, and the like. The composition can be formulated as a suppository with conventional binding agents and carriers such as triglycerides. The oral preparation may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, pharmaceutical grade magnesium carbonate, etc. Examples of approaches

- 17 045759 active pharmaceutical carriers are described in the source Remington's Pharmaceutical Sciences per ed. E. W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide a form for proper administration to a patient. The drug must correspond to the method of administration. The parenteral drug may be contained in ampoules, single-use syringes, or multi-dose vials made of glass or plastic.

In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in a sterile isotonic aqueous buffer solution. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to relieve pain at the injection site. Typically, the ingredients are supplied either individually or as a mixture in a unit dosage form, such as a dry lyophilized powder or an anhydrous concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active agent. If the composition is to be administered by infusion, it may be placed, for example, in an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

The compounds according to the invention can be included in the preparation in the form of neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions, such as salts formed with hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and salts formed with cations, such as salts formed with sodium, potassium, ammonium , calcium, iron (III) hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Examples

Example 1. Preparation of mouse monoclonal antibodies to human SIRPa

Human SIRPa protein was used to immunize various strains of mice, and hybridomas were generated accordingly. Eight fusions were made to obtain a sufficient number of hybridoma clones. SIRPa v1/v2-positive binders were selected and subcloned. In vitro binding and functional screening was then performed using approximately 30 purified antibodies and the best antibodies with the highest binding affinity and the strongest functional activity were identified. The best antibodies have been humanized.

The VH/VL sequences of the best mouse antibodies are presented in the table below.

Table 1. VH/VL sequence of the best mouse antibodies

Name Sequence (CDRs underlined) SEQ ID NO: 248G3F6 VH EVQLQQSGAELVKPGASVKLSCTASGFNFEDTYMHWVKQRPDQGLEWIGR IDPADGDTKYNPKFQDKATITVDT S SNTAYLQLS S LT S EDTAVYYCVRGN YVNWGQGTTLTVSS 1 248G3F6 VL QIVLIQSPAIMSASРGERVTLTCRASSSVSSSYLYWYQQКРGSSPKLWIY STSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWYSYPRTFG GGTKLEIK 2 300Ab Ab VH QVQLQQSGTELVKPGSSVKISCKASGYTFTSNYIHWIRQQPGNGLEWIGW IYPGDGDTNYNQKFNGKATLTADKS S STAYMQLS SLTS EDYAVYFCAINY GGIWFAYWGQ GT LVTVS S 3 300Ab Ab VL DIQMTQSPSSSMSASLGDRVTITCQASQDIGNKLIWFQQKPGKSPRLMIHY VTNLPGGVPLRFSGSRSGSDYSLTISSLESEDMADYYCLQYKQNPLTFGS GTKLEIK 4 102A10F2 VH QVTLKESGPGILQPSQTLSLTCSFSGFSLNTYDIGMGWIRQPSGKGLEWL AHIWWNDREYYNSALQSRVTISKDTSNTQVFLKIASVDTADTATYYCVRI DYFGSGQAWFTYWGQGTLVTVSA 5 102A10F2 VL EIVLTQSPPTMAASPGEKITITCSSSSTISSTYLHWYQQKPGFSPKLLIS GTSNLASGVPPRFSGSGSGTSYSLTIGTLEAEDVATYYCQQGSRIPFTFG SGTKLEIK 6

- 18 045759

62D2H6 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWVKQRTEQGLEWIGR IDPEDGETKYAPKFQGKATITADT S SNTAYLQLS S LTS EDTAVYYC S RSW AYWGQGTTLTVSS 7 62D2H6 VL QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPKLWIY STSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPRTFG GGTKLEIK 8 211F8E11 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWVGR IDPANVNTIYDPKFQGKATITADT S SNTAYLQLS SLTSEDTAVYYCARVG AYDGYDFDYWGQGTTLTVSS 9 211F8E11 VL DIVLTQS PASLAVS LGQRATIS CRASESVDNYGNSFMHWYQQKPGQPPKL LIYRASNLESGI PARE S G S G S RT D FT LTIN PVEADDVATYYCQQNNEDPL TFGAGTKLELK 10 217D11E5 VH EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGY INPYNDGTKYNEKFKGKATLTSDKSS STAYMELS SLTSEDSAVYYCARSY YDYDGSFDYWGQGTTLTVSS eleven 217D11E5 VL DIVMTQSHKFMSTSVGDRVSITCKASQDVTTAVAWYQQKSGQSPKLLIYS ASYRYTGDPDRFTGSGSGTDFTFTIS SVQAED LAVYYCQQHYSTPWT FG G GTKLEIK 12 234B7D5 VH EVQLQQSGAELVKPGASVKLSCTASGFNFEDTYIHWVKQRPDQGLEWIGR IDPADGDTKHNPKFHDKATVTVDTS SNTAYLELS S LTS EDTAVYYCVRGN YVNWGQGTTLTVSS 13 234B7D5 VL QIVLIQSPAIMSASPGERVTLTCRASSSVTSSYLYWYQQKPG5SPKLWIY SASNLASGVPARFSGSGSGTSYSLTISSVEAEDAASYFCHQWYSYPRTFG GGTKLEIK 14

Example 2: Crosslinking SIRPa v1 and v2

In this example, the dose-dependent binding response of a mouse anti-SIRPa mAb to recombinant human SIRPa variant 1 and variant 2 protein (0.5 μg/ml in a 100 μl dose) was measured in an ELISA. Recombinant human SIRPa v1 or v2 protein (Biointron) was applied at a concentration of 0.5 μg/ml in PBS to microtiter plates for 2 h at room temperature. After antigen application, wells were blocked with PBS/0.05% Tween (PBST) with 1% BSA for 1 h at room temperature.

After washing the wells with PBST, various concentrations of anti-SIRPa antibodies were added to the wells and incubated for 1 h at room temperature. To detect binding antibodies, HRP-conjugated anti-mouse Fc secondary antibodies (Jackson Immuno Research) were added followed by fluorogenic substrates (Roche). Between all incubation steps, the plate wells were washed three times with PBST. Fluorescence was measured in a TECAN Spectrafluor plate reader.

The results are shown in Fig. 1. Both antibodies tested, 248G3F6 and 300A6A6, demonstrated nanogram-level affinity for both variants 1 and 2.

Antibody binding kinetics analysis of variant 1 was performed using the Biacore 8K system using a human antibody capture approach. Anti-mouse Fc IgG was immobilized on the CM5 sensor chip according to the manufacturer's instructions. The antibody of interest was injected and captured by immobilized anti-human Fc IgG. Separately, successive concentrations of antigen were administered and the binding profile for the analyte-antigen at each concentration was recorded, respectively.

The analytical system was regenerated by introducing 10 mM glycine-HCl, pH 1.5, for 30 s. The running buffer was HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% P20). The assay temperature was 25°C, and the association and dissociation times were 180 and 600 s, respectively. Biacore data were fitted using Biacore K8 1.0 evaluation software according to a 1:1 binding model to calculate association (ka) and dissociation (kd) rate constants as well as equilibrium constant (KD).

The results are shown in Fig. 2 and summarized in the table below. Both antibodies tested showed excellent binding affinity.

Sample ka (1/Mc) kd(1/c) KD(M) 248G3F6 l,87E+05 3.74E-04 2.00E-09 300A6A6 l,31E+05 l,48E-04 1DZE-09

Example 3: Competition with CD47

In this example, the ability of anti-SIRPa antibodies to compete with CD47 for binding to SIRPa was examined.

Recombinant CD47-Fc fusion protein (Acrobiosystems) was applied at a concentration of 1 μg/ml in PBS to microtiter plates for 16 h at 4°C. After blocking for 1 h with 1% BSA in PBST at room temperature, 1 μg/ml SIRPa-His protein was added either in the absence or presence of various concentrations of anti-SIRPa antibodies at room temperature for 1 h. The plates were then washed three times and incubated with HRP -conjugated secondary antibody to His for 1 hour at room temperature. After washing, TMB solution was added to each well for 30 min, the reaction was stopped with 2M H2SO4, and OD (optical density) was measured at 490 nm.

As shown in FIG. 3, both 248G3F6 and 300A6A6 potently and dose-dependently inhibited CD47 binding to SIRPa.

Example 4: Induction of Macrophage-Mediated Phagocytosis

- 19 045759

In this example, the ability of anti-SIRPa antibodies to induce macrophage-mediated phagocytosis was examined.

PBMCs (peripheral blood mononuclear cells) were isolated from human blood and monocytes were differentiated into macrophages within 6 days. Monocyte-derived macrophages (MDM) were scraped and replated into 24-well plates and allowed to adhere for 24 hours. The human tumor cell line Raji, which endogenously expressed CD47, was transfected with human PD-L1 to overexpress human PD-L1 on the surface. These PD-L1-overexpressing Raji cells were selected as target cells and labeled with 1 μM CFSE for 10 min, then added to MDM at a ratio of 5:1 tumor cells per phagocyte.

Anti-SIRP-alpha antibody and anti-PD-L1 antibody were added to the culture system. After incubation for 3 h, nonphagocytosed target cells were washed with PBS, and remaining phagocytes were scraped off, stained with macrophage marker antibody C76, and analyzed by flow cytometry. Phagocytosis was measured by gating on C76 ⁺ cells and then assessing the percentage of CFSE+ cells.

The results of phagocytosis of tumor cells expressing PD-L1 by combined treatment with anti-SIRPa antibody and anti-PD-L1 antibody are shown in FIG. 4. The combination of anti-PD-L1 antibody with any of the anti-SIRPa antibodies demonstrated the highest phagocytosis (two columns on the right).

Example 5 Humanization of Murine mAbs

Mouse antibody variable region genes were used to create humanized mAbs. In the first step of this process, the amino acid sequences of the VH and VL mAbs were compared to an available database of human Ig gene sequences to find the best matching human Ig germline gene sequences.

The amino acid sequences of the humanized antibody are presented below.

Humanized sequences of A. 248G3F6

Table 2A. Humanization 248G3F6-VH

Name Subsequence SEQ ID NO: 248G3F6 VH EVQLQQSGAELVKPGASVKLSCTASGFNFEDTYMHWVKQRPDQGLEWIGR IDPADGDTKYNPKFQDKATITVDT S SNTAYLQLS S LT S EDTAVYYCVRGN YVNWGQGTTLTVSS 1 VI (CDR grafting) QVQLVQSGAEVKKPGASVKVSCKASGFNFEDTYMHWVRQAPGQGLEWMGR IDPADGDTKYNPKFQDRVTMTRDT S T S T VYME L S S L RS EDTAVYYCARGN YVNWGQGTTVTVSS 23 V2 (with back mutations) QVQLVQSGAEVKKPGASVKVSCKASGFNFEDTYMHWVRQAPGQGLEWMGR IDPADGDTKYNPKFQDRVTMTVDTSTNTAYMELSSLRSEDTAVYYCVRGN YVNWGQGTTVTVSS 24 V3 (with back mutations) QVQLVQSGAEVKKPGASVKVSCKASGFNFEDTYMHWVRQAPGQGLEWMGR IDPADGDTKYNPKFQDRVTITVDTSTNTAYMELSSLRSEDTAVYYCVRGN YVNWGQGTTVTVSS 25 V4 (with back mutations) QVQLVQSGAEVKKPGASVKVSCKASGFNFEDTYMHWVRQAPGQGLEWMGR IDPAEGDTKYNPKFQDRVTITVDTSTNTAYMELSSLRSEDTAVYYCVRGN YVNWGQGTTVTVSS 26 V5 (with back mutations) QVQLVQSGAEVKKPGASVKVSCKASGFNFEDTYMHWVRQAPGQGLEWMGR IDPADADTKYNPKFQDRVTITVDTSTNTAYMELSSLRSEDTAVYYCVRGN YVNWGQGTTVTVSS 27

Table 2B. CDR sequences

CDR Subsequence SEQ ID NO: CDR-H1 DTYMH 15 CDR-H2 RIDPADGDTKYNPKFQD 16 CDR-H3 GNYVN 17 CDR-H2 (v4) RIDPAEGDTKYNPKFQD 21 CDR-H2 (v5) RIDPADADTKYNPKFQD 22

Table 2C. Humanization 248G3F6-VL

Name Subsequence SEQ ID NO: 248G3F6 VL QIVLIQSPAIMSASPGERVTLTCRASSSVSSSYLYWYQQKPGSSPKLWIY STSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWYSYPRTFG GGTKLEIK 2 VI (CDR grafting) EIVLTQSPGTLLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQAPRLLIY STSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQWYSYPRTFG GGTKVEIK 28 V2 (with back mutations) EIVLTQSPGTLLSLSPGERATLSCRASSSVSSSYLYWYQQKPGQAPRLLIY STSNLASGIPDRFSGSGSGTDYTLTISRLEPEDAAVYFCHQWYSYPRTFG GGTKVEIK 29

Table 2D. CDR sequences

CDR Subsequence SEQ ID NO: CDR-L1 RASSSVSSSYLY 18 CDR-L2 STSNLAS 19 CDR-L3 HQWYSYPRT 20

- 20 045759

Table 2E. Humanized Antibodies

VL VLvl VLv2 VH HSP210-02-Chi VHvl HSP210-02-hzll HSP210-02-hzl2 VHv2 HSP210-02-hz21 HSP210-02-hz22 VHv3 HSP210-02-hz31 HSP210-02-hz32 VHv4 HSP210-02-hz41 HSP210-02-hz42 VHv5 HSP210-02-hz51 HSP210-02-hz52

V. 300A6A6

Table 3A. Humanization 3OOA6A6-VH

Name Subsequence SEQ ID NO: 300A6A6 VH QVQLQQSGTELVKPGSSVKISCKASGYTFTSNYIHWIRQQPGNGLEWIGW IYPGDGDTNYNQKFNGKATLTADKSSSTAYMQLSSLTSEDYAVYFCAINY GGIWFAYWGQGTLVTVS S 3 VI (CDR grafting) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSNYIHWVRQAPGQGLEWMGW IYPGDGDTNYNQKFNGRVTITADKST STAYMELS SLRSEDTAVYYCARNY GGIWFAYWGQGTLVTVSS 39 V2 (with back mutations) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSNYIHWVRQAPGQGLEWMGW IYPGDGDTNYNQKFNGRVTITADKSTSTAYMELSSLRSEDTAVYYCAINY GGIWFAYWGQGTLVTVSS 40 V3 (with back mutations) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSNYIHWVRQAPGQGLEWMGW IYPGDGDTNYNQKFNGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAINY GGIWFAYWGQGTLVTVSS 41 V4 (with back mutations) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSNYIHWVRQAPGQGLEWMGW IYPGEGDTNYNQKFNGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAINY GGIWFAYWGQGTLVTVSS 42 V5 (with back mutations) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSNYIHWVRQAPGQGLEWMGW IYPGDADTNYNQKFNGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAINY GGIWFAYWGQGTLVTVSS 43 V6 (with back mutations) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSNYIHWVRQAPGQGLEWMGW IYPGDGDTNYNQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAINY GGIWFAYWGQGTLVTVSS 44

Table 3B. CDR sequences

CDR Subsequence SEQ ID NO: CDR-H1 SNYIH thirty CDR-H2 WIYPGDGDTNYNQKFNG 31 CDR-H3 NYGGIWFAY 32 CDR-H2 (v4) WIYPGEGDTNYNQKFNG 36 CDR-H2 (v5) WIYPGDADTNYNQKFNG 37 CDR-H2 (v6) WIYPGDGDTNYNQKFQG 38

Table 3C. Humanization 300A6A6-VL

Name Subsequence SEQ ID NO: 300A6A6 VL DIQMTQSPSSSMSASLGDRVTITCQASQDIGNKLIWFQQKPGKSPRLMIHY VTNLPGGVPLRFSGSRSGSDYSLTISSLESEDMADYYCLQYKQNPLTFGS GTKLEIK 4 VI (CDR grafting) DIQMTQSPSSSLSASVGDRVTITCQASQDIGNKLIWYQQKPGKAPKLLIYY VTNLPGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYKQNPLTFGQ GTKLEIK 45 V2 (with back mutations) DIQMTQSPSSSLSASVGDRVTITCQASQDIGNKLIWFQQKPGKAPKLLIHY VTNLPGGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYKQNPLTFGQ GTKLEIK 46

Table 3D. CDR sequences

CDR Subsequence SEQ ID NO: CDR-L1 QASQDIGNKLI 33 CDR-L2 YVTNLPG 34 CDR-L3 LQYKQNPLT 35

Table 3E. Humanized Antibodies

VL VLvl VL v2 VH HSP210-03-Chi VHvl HSP210-03-hzll HSP210-03-hzl2 VHv2 HSP210-03-hz21 HSP210-03-hz22 VHv3 HSP210-03-hz31 HSP210-03-hz32 VHv4 HSP210-03-hz41 HSP210-03-hz42 VHv5 HSP210-03-hz51 HSP210-03-hz52 VHv6 HSP210-03-hz61 HSP210-03-hz62

S. 102A10F2

Table 4A. Humanization 102A10F2-VH

Name Subsequence SEQ ID NO: 102A10F2 VH QVTLKESGPGILQPSQTLSLTCSFSGFSLNTYDIGMGWIRQPSGKGLEWLAH IWWNDREYYNSALQSRVTI5KDT5MTQVFLKIASVDTADTATYYCVRIDYFG SGQAWFTYWGQ GT LVTVSA 5 VI (CDR grafting) QLQLQESGPGLVKPSETLSLTCTVSGFSLNTYDIGMGWIRQPPGKGLEWIGH IWWNDREYYNSALQSRVTISVDT S KNQFS LKLS SVTAADTAVYYCARIDYFG SGQAWFTYWGQ GT LVTVS S 55

- 21 045759

V2 (with back mutations) QVQLQESGPGLVKPSETLSLTCTFSGFSLNTYDIGMGWIRQPPGKGLEWIGH IWWNDREYYNSALQSRVTISKDTSKTQVSLKLSSVTAADTAVYYCVRIDYFG SGQAWFTYWGQGTLVTVSS 56 V3 (with back mutations) QVQLQESGPGLVKPSETLSLTCTFSGFSLNTYDIGMGWIRQPPGKGLEWIAH IWWNDREYYNSALQSRVTISKDTSKTQVSLKLSSVTAADTAVYYCVRIDYFG SGQAWFTYWGQGTLVTVSS 57 V4 (with back mutations) QVQLQESGPGLVKPSETLSLTCTFSGFSLSTYDIGMGWIRQPPGKGLEWIAH IWWNDREYYNSALQSRVTISKDTSKTQVSLKLSSVTAADTAVYYCVRIDYFG SGQAWFTYWGQGTLVTVSS 58 V5 (with back mutations) QVQLQESGPGLVKPSETLSLTCTFSGFSLNTYDIGMGWIRQPPGKGLEWIAH IWWNDREYYSSALQSRVTISKDTSKTQVSLKLSSVTAADTAVYYCVRIDYFG SGQAWFTYWGQGTLVTVSS 59 V6 (with back mutations) QVQLQESGPGLVKPSETLSLTCTFSGFSLNTYDIGMGWIRQPPGKGLEWIAH IWWNDREYYNPALQSRVTISKDTSKTQVSLKLSSVTAADTAVYYCVRIDYFG SGQAWFTYWGQGTLVTVSS 60

Table 4B. CDR sequences

CDR Subsequence SEQ ID NO: CDR-H1 TYDIGMG 47 CDR-H2 HIWWNDREYYNSALQS 48 CDR-H3 IDYFGSGQAWFTY 49 CDR-H2 (v5) HIWWNDREYYSSALQS 53 CDR-H2 (v6) HIWWNDREYYNPALQS 54

Table 4C. Humanization 102A10F2-VL

Name Subsequence SEQ ID NO: 102A10F2 VL EIVLTQSPPTMAASPGEKITITCSSSSTISSTYLHWYQQKPGFSPKLLIS STSNLASGVPPRFSGSGSGTSYSLTIGTLEAEDVATYYCQQSSRIPFTFG SGTKLEIK 6 VI (CDR grafting) EIVLTQSPGTLLSLSPGERATLSCSSSSTISSTYLHWYQQKPGQAPRLLIY GTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGSRIPFTFG QGTKLEIK 61 V2 (with back mutations) EIVLTQSPGTLLSLSPGERATLSCSSSSTISSTYLHWYQQKPGQAPRLLIS GTSNLASGIPDRFSGSGSGTDYTLTISRLEPEDVAVYYCQQGSRIPFTFG QGTKLEIK 62

Table 4D. CDR sequences

CDR Subsequence SEQ ID NO: CDR-L1 SSSSTISSTYLH 50 CDR-L2 GTSNLAS 51 CDR-L3 QQGSRIPFT 52

Table 4E. Humanized Antibodies

VL VLvl VL v2 VH HSP210-01-Chi VHvl HSP210-01-hzll HSP210-01-hzl2 VHv2 HSP210-01-hz21 HSP210-01-hz22 VHv3 HSP210-01-hz31 HSP210-01-hz32 VH v4 HSP210-01-hz41 HSP210-01-hz42 VHv5 HSP210-01-hz51 HSP210-01-hz52 VHv6 HSP210-01-hz61 HSP210-01-hz62

D.62D2H6

Table 5A. Humanization 62D2H6-VH

Name Subsequence SEQ ID NO: 62D2H6 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWVKQRTEQGLEWIGR IDPEDGETKYAPKFQGKATITADTS SNTAYLQLS S LT S EDTAVYYCSRSW AYWGQGTTLTVSS 7 VI (CDR grafting) EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYMHWVQQAPGKGLEWMGR IDPEDGETKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATSW AYWGQGTTVTVSS 69 V2 (with back mutations) EVQLVQSGAEVKKPGATVKISCKASGFNIKDYYMHWVQQAPGKGLEWMGR IDPEDGETKYAPKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCSRSW AYWGQGTTVTVSS 70 V3 (chimeric 2) QVQ LVQ S GAEVKK P GASVKVS OKAS G FNIKDYYMHWVRQAPGQGL EWMGR IDPEDGETKYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSW AYWGQGTTVTVSS 71 V4 (with back mutations) QVQ LVQ S GAEVKK P GASVKVS OKAS G FNIKDYYMHWVRQAP GQGL EWMG R IDPEDGETKYAPKFQGRVTMTADTSTNTAYMELSSLRSEDTAVYYCSRSW AYWGQGTTVTVSS 72

Table 5B. CDR sequences

CDR Subsequence SEQ ID NO: CDR-H1 DYYMH 63 CDR-H2 RIDPEDGETKYAPKFQG 64 CDR-H3 SWAY 65

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Table 5C. Humanization 62D2H6-VL

Name Subsequence SEQ ID NO: 62D2H6 VL QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPKLWIYST SNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPRTFGGGTK LEIK 8 VI (CDR grafting) EIVLTQSPATLSLSPGERATLSCSASSSVSSSYLYWYQQKPGQAPRLLIYST SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQWSSYPRTFGGGTK VEIK 73 V2 (with back mutations) EIVLTQSPATLSLSPGERATLSCSASSSVSSSYLYWYQQKPGQAPRLLIYST SNLASGIPARFSGSGSGTDYTLTISSLEPEDAAVYFCHQWSSYPRTFGGGTK VEIK 74 V3 (chimeric 2) EIVMTQSPPTLSLSPGERVTLSCSASSSVSSSYLYWYQQKPGQAPRLLIYST SNLASGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCHQWSSYPRTFGGGTK VEIK 75 V4 (with back mutations) EIVMTQSPPTLSLSPGERVTLSCSASSSVSSSYLYWYQQKPGQAPRLLIYST SNLASGIPARFSGSGSGTDYTLTISSLQPEDAAVYFCHQWSSYPRTFGGGTK VEIK 76

Table 5D. CDR sequences

CDR Subsequence SEQ ID NO: CDR-L1 SASSSVSSSYLY 66 CDR-L2 STSNLAS 67 CDR-L3 HQWSSYPRT 68

Table 5E. Humanized Antibodies

VL VLvl VLv2 VLv3 VLv4 VH Chimeric VHvl hzll VHv2 hz22 hz24 VHv3 hz33 VHv4 hz42 hz44

E. 211F8E11

Table 6A. Humanization 211F8E11-VH

Name Subsequence SEQ ID NO: 211F8E11 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWVGR IDPANVNTIYDPKFQGKATITADT S SNTAYLQL S S LT S EDTAVYYCARVG AYDGYDFDYWGQGTTLTVS S 9 VI (CDR grafting) EVQLVQSGAEVKKPGATVKISCKVSGFNIKDTYMHWVQQAPGKGLEWMGR IDPANVNTIYDPKFQGRVTITADT S T DTAYMEL S S LRS EDTAVYYCATVG AYDGYDFDYWGQGTTVTVSS 83 V2 (with back mutations) EVQLVQSGAEVKKPGATVKISCKASGFNIKDTYMHWVQQAPGKGLEWMGR IDPANVNTIYDPKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCARVG AYDGYDFDYWGQGTTVTVSS 84 V3 (chimeric 2) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYMHWVRQAPGQGLEWMGR IDPANVNTIYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVG AYDGYDFDYWGQGTTVTVSS 85 V4 (with back mutations) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYMHWVRQAPGQGLEWMGR IDPANVNTIYDPKFQGRVTMTADTSTNTAYMELSSLRSEDTAVYYCARVG AYDGYDFDYWGQGTTVTVSS 86 Name Table 6B. CDR sequences CDR Sequence SEQ ID NO: CDR-H1 DTYMH 77 CDR-H2 RIDPANVNTIYDPKFQG 78 CDR-H3 VGAYDGYDFDY 79 Table 6C. Humanization 211F8E11-VL Subsequence SEQ ID NO: | 211F8E11 VL DIVLTQS PAS LAVS LGQRATIS CRASESVDNYGNSFMHWYQQKPGQPPKLLI YRASNLESGIPAR FS G S G S RTD FT LTIN PVEADDVATYYCQQNNEDPLTFGA GTKLELK 10 VI (CDR grafting) DIVMTQSPDSLAVSLGERATINCRASESVDNYGNSFMHWYQQKPGQPPKLLI YRASNLESGVPDR FSGSGSGTDFTLTIS SLQAEDVAVYYCQQNNEDPLTFGQ GTKLEIK 87 V2 (with back mutations) DIVLTQSPDSLAVSLGERATINCRASESVDNYGNSFMHWYQQKPGQPPKLLI YRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPLTFGQ GTKLEIK 88 V3 (chimeric 2) DIQMTQSPSSLSSASVGDRVTITCRASESVDNYGNSFMHWYQQKPGKVPKLLI YRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQNNEDPLTFGQ GTKLEIK 89 V4 (with back mutations) DIQLTQSPSSSLSASVGDRVTITCRASESVDNYGNSFMHWYQQKPGKVPKLLI YRASNLESGVPSRFSGSGSRTDFTLTISSLQPEDVATYYCQQNNEDPLTFGQ GTKLEIK 90

Table 6D. CDR sequences

CDR Subsequence SEQ ID NO: CDR-L1 RASESVDNYGNSFMH 80 CDR-L2 RASNLES 81 CDR-L3 QQNNEDPLT 82

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Table 6E. Humanized Antibodies

VL VLvl VLv2 VLv3 VLv4 VH Chimeric VHvl hzll VHv2 hz22 hz24 VHv3 hz33 VHv4 hz42 hz44

F.217D11E5

Table 7A. Humanization 217D11E5-VH

Name Subsequence SEQ ID NO: 217D11E5 VH EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGY INPYNDGTKYNBKFKGKATLTSDKSSS T AYME L S S LT S ED SAVYYCARSY YDYDGSFDYWGQGTT LTVSS eleven VI (CDR grafting) QVQ LVQS GAEVKKP GASVKVS C KAS GYT FT SYVMHWVRQAP GQRLEWMGY INPYNDGTKYNBKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARSY YDYDGSFDYWGQGTTVTVSS 97 V2 (with back mutations) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGY INPYNDGTKYNEKFKGRVTITSDKSASTAYMELSSLRSEDTAVYYCARSY YDYDGSFDYWGQGTTVTVS S 98 V3 (chimeric 2) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGY INPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSY YDYDGS FDYWGQGTTVTVS S 99 V4 (with back mutations) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGY INPYNDGTKYNEKFKGRVTMTSDKSTSTAYMELSSLRSEDTAVYYCARSY YDYDGSFDYWGQGTTVTVSS 100

Table 7B. CDR sequences

CDR Subsequence SEQ ID NO: CDR-H1 SYVMH 91 CDR-H2 YINPYNDGTKYNEKFKG 92 CDR-H3 SYYDYDGSFDY 93

Table 7C. Humanization 217D11E5-VL

Name Subsequence SEQ ID NO: 217D11E5 VL DIVMTQSHKFMSTSVGDRVSITCKASQDVTTAVAWYQQKSGQSPKLLIYSAS YRYTGDPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKL EIK 12 VI (CDR grafting) DIQMTQSPSSLSASVGDRVTITCKASQDVTTAVAWYQQKPGKARKLLIYSAS YRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPWTFGGGTKV EIK 101 V2 (chimeric 2) DIQMTQSPSSLSASVGDRVTITCKASQDVTTAVAWYQQKPGKVPKLLIYSAS YRYTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQHYSTPWTFGGGTKV EIK 102

Table 7D. CDR sequences

CDR Subsequence SEQ ID NO: CDR-L1 KASQDVTTAVA 94 CDR-L2 SASYRYT 95 CDR-L3 QQHYSTPWT 96

Table 7E. Humanized Antibodies

VL VLvl VLv2 VH Chimeric VHvl hzll VHv2 hz21 hz22 VHv3 VHv4 hz41 hz42

G. 234B7D5

Table 8A. Humanization 234B7D5-VH

Name Subsequence SEQ ID NO: 234B7D5 VH EVQLQQSGAELVKPGASVKLSCTASGFNFEDTYIHWVKQRPDQGLEWIGRID PADGDTKHNPKFHDKATVTVDTSSNTAYLELSSLTSEDTAVYYCVRGNYVNW 13 GQGTTLTVSS VI (CDR grafting) EVQLVQSGAEVKKPGATVKISCKVSGFNFEDTYIHWVQQAPGKGLEWMGRID PADGDTKHNPKFHDRVTITADT S T DTAYME L S S L RS EDTAVYYCATGNYVNW 109 GQGTTVTVSS V2 (with back mutations) EVQLVQSGAEVKKPGATVKISCKASGFNFEDTYIHWVQQAPGKGLEWMGRID PADGDTKHNPKFHDRVTITVDTSTNTAYMELSSLRSEDTAVYYCVRGNYVNW GQGTTVTVSS 110 V3 (chimeric 2) QVQLVQSGAEVKKPGASVKVSCKASGFNFEDTYIHWVRQAPGQGLEWMGRID PADGDTKHNPKFHDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGNYVNW GQGTTVTVSS 111 V4 (with back mutations) QVQLVQSGAEVKKPGASVKVSCKASGFNFEDTYIHWVRQAPGQGLEWMGRID PADGDTKHNPKFHDRVTMTVDTSTNTAYMELSSLRSEDTAVYYCVRGNYVNW GQGTTVTVSS 112

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Table 8B. CDR sequences

CDR Subsequence SEQ ID NO: CDR-H1 DTYIH 103 CDR-H2 RIDPADGDTKHNPKFHD 104 CDR-H3 GNYVN 105

Table 7C. Humanization 234B7D5-VL

Name Subsequence SEQ ID NO: 234B7D5 VL QIVLIQSPAIMSASPGERVTLTCRASSSVTSSYLYWYQQKPGSSPKLWIYSA SNLASGVPARFSGSGSGTSYSLTISSVEAEDAASYFCHQWYSYPRTFGGGTK LEIK 14 VI (CDR grafting) EIVLTQSPATLSLSPGERATLSCRASSSVTSSYLYWYQQKPGQAPRLLIYSA SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQWYSYPRTFGGGTK VEIK 113 V2 (with back mutations) EIVLTQSPATLSLSPGERATLSCRASSSVTSSYLYWYQQKPGQAPRLLIYSA SNLASGIPARFSGSGSGTDYTLTISSLEPEDAAVYFCHQWYSYPRTFGGGTK VEIK 114 V3 (chimeric 2) EIVLTQSPGTLLSLSPGERATLSCRASSSVTSSYLYWYQQKPGQAPRLLIYSA SNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQWYSYPRTFGGGTK VEIK 115 V4 (with back mutations) EIVLTQSPGTLLSLSPGERATLSCRASSSVTSSYLYWYQQKPGQAPRLLIYSA SNLASGIPDRFSGSGSGTDYTLTISRLEPEDAAVYFCHQWYSYPRTFGGGTK VEIK 116 V5 (chimera 3) EIVMTQSPPTLSLSPGERVTLSCRASSSVTSSYLYWYQQKPGQAPRLLIYSA SNLASGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCHQWYSYPRTFGGGTK VEIK 117 V6 (with back mutations) EIVMTQSPPTLSLSPGERVTLSCRASSSVTSSYLYWYQQKPGQAPRLLIYSA SNLASGIPARFSGSGSGTDYTLTISSLQPEDAAVYFCHQWYSYPRTFGGGTK VEIK 118

Table 8D. CDR sequences

CDR Subsequence SEQ ID NO: CDR-L1 RASSSVTSSYLY 106 CDR-L2 SASNLAS 107 CDR-L3 HQWYSYPRT 108

Table 8E. Humanized Antibodies

VL VLvl VLv2 VLv3 VLv4 VLv5 VLv6 VH Chimeric VHvl hzll VHv2 hz22 hz24 hz26 VHv3 hz33 VHv4 hz42 hz44 hz46

Example 6 Testing Humanized Antibodies

In this example, some of the humanized antibodies were tested for the ability to block interactions between SIRPa and CD47.

Recombinant CD47-Fc fusion protein (Acrobiosystems) was applied at a concentration of 1 μg/ml in PBS to microtiter plates for 16 h at 4°C. After blocking for 1 h with 1% BSA in PBST at room temperature, 1 μg/ml SIRPa-His protein was added either in the absence or presence of various concentrations of anti-SIRPa antibodies at room temperature for 1 h. The plates were then washed three times and incubated with HRP -conjugated secondary antibody to His for 1 hour at room temperature. After washing, TMB solution was added to each well for 30 minutes, the reaction was stopped with 2M H ₂ SO ₄ and OD was measured at 490 nm.

All antibodies listed in Tables 2E (248G3F6), 3E (300A6A6) and 4E (102A10F2) were tested and demonstrated high IC50 values (Table 9).

Table 9. Activity of humanized antibodies in blocking the interaction of SIRPa with CD47

Antibody IC50 (nM) 248G3F6 02-chi 0.14 02-hz22 0.092 02-hz32 0.11 02-hz42 0.12 02-hz52 0.11 300A6A6 03-chi 0.14 03-hz22 0.16 03-hz32 0.145 03-hz42 0.13 03-hz52 0.13 102A10F2 01-hz22 0.21 01-hz32 0.16 01-hz52 0.23 01-hz61 0.21 01-hz62 0.16

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Example 7: Enhancing Macrophage-Mediated Phagocytosis of Tumor Cells

In this example, some of the humanized antibodies were tested for their ability to enhance macrophage-mediated phagocytosis of tumor cells.

PBMCs were isolated from human blood and monocytes were differentiated into macrophages using a standard protocol. Monocyte-derived macrophages (MDM) were scraped and replated into 24-well plates and allowed to adhere for 24 hours. The Raji human tumor cell line, which endogenously expressed CD47, was selected as target cells and labeled with 1 μM CFSE for 10 minutes. , were then added to MDM at a ratio of 5:1 tumor cells per phagocyte, and various concentrations of anti-SIRPa antibodies were added at the indicated concentrations. After 3 h of incubation, nonphagocytosed target cells were washed with PBS, and the remaining phagocytes were scraped off, stained with C76 antibody, and analyzed by flow cytometry. Phagocytosis was measured by gating on C76 ⁺ cells and then assessing the percentage of CFSE+ cells.

The results are presented in Fig. 5. Of the antibodies tested, 02-hz52 (248G3F6) and 03-hz51 (300A6A6) showed the greatest activity, and all others also showed excellent activity.

Example 8: Binding affinity for SIRPa v1 and v2

In this example, humanized antibodies 02-hz52 (248G3F6) and 03-hz51 (300A6A6) were tested for their binding affinity to SIRPa v1 and v2.

Antibody-antigen binding kinetics analysis was performed using the Biacore 8K system using a human antibody capture approach. Anti-mouse Fc IgG was immobilized on the CM5 sensor chip according to the manufacturer's instructions. The antibody of interest was injected and captured by immobilized anti-human Fc IgG. Successive concentrations of human SIRPa v1 or SIRPa v2 protein were then administered individually, and the binding profile for the antigen analyte at each concentration was recorded, respectively. The analytical system was regenerated by introducing 10 mM glycine-HCl, pH 1.5, for 30 s. The running buffer was HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% P20). The assay temperature was 25°C, and the association and dissociation times were 180 and 600 s, respectively. Biacore data were fitted using Biacore K8 1.0 evaluation software according to a 1:1 binding model to calculate association (ka) and dissociation (kd) rate constants as well as equilibrium constant (KD).

The test results are shown in table. 10A-B.

Table 10A. Binding affinity for SIRPa v1

Sample ka (1/Ms) kd (1/s) KD (M) 02-hz52 7.04E+05 2.98E-04 4.23E-10 03-hz51 8D5E+05 8D4E-04 9.99E-10

Table 10B. Binding affinity for SIRPa v2

Sample ka (1/Ms) kd (1/s) KD (M) 02-hz52 3,ЗЗЭ+О5 2.47E-03 7.40E-09 03-hz51 3.31E+05 1.00E-03 3, OZE-09

Example 9: Manufacturing feasibility studies

Antibodies 02-hz52 (248G3F6) and 03-hz51 (300A6A6), fused to human IgG4 heavy chain constant regions and kappa light chain constant regions, were tested for their suitability for production. Both antibodies do not contain free cysteines. Test results showed that both antibodies could be purified to clinical purity and had suitable Tm, acid/base peak ratios, hydrophobicity and pI. The results are presented in table. eleven.

Table 10A. Binding affinity for SIRPa v1

Sample Aggregates SEC (% monomer) Yield (mg/l) pl Acid PEAK Basic PEAK Main PIC Tm pH 7.5 (°C) HIC (NlhhSOj (Μ) 02-hz52 Not detected 99.2 >10 6.9 35.5 57.2 7.2 65/71 1.02 03-hz51 Not detected 97.91 >10 7.9 34.5 62.3 3.2 65/74 0.96

Example 10 In Vivo Study

In this example, the effectiveness of the humanized antibody 03-hz51 (300A6A6) in mice, alone or in combination with rituximab, was examined.

Raji-Luc cells resuspended in PBS were inoculated into the tail vein of humanized B-NDG-hSIRPa mice at a concentration of 1x105 cells/0.2 ml and in a volume of 0.2 ml/animal. A small animal imaging device was used to measure the tumor imaging signal value on the third day of inoculation. When the average imaging signal intensity reached 1.35 x 106 fps, the animals were divided into groups according to the tumor imaging signal value and the animal's body weight and were evenly distributed across 4 experiments. There were 6 mice in each experimental group.

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Rituximab demonstrated significant inhibitory effects on Raji-Luc lymphoma at the 10 mg/kg dose level, without clinically adverse symptoms. 03-hz51 in combination with rituximab (10 mg/kg + 0.1 mg/kg) demonstrated significant inhibitory effects on Raji-Luc lymphoma. Data show that 03-hz51 interacted synergistically with rituximab to cause complete inhibition of tumor growth in a Raji lymphoma model.

The scope of the present invention is not to be limited by the individual embodiments described, which serve as individual illustrations of specific aspects of the invention, and any compositions or methods that are functionally equivalent are within the scope of the present invention. Those skilled in the art will appreciate that the methods and compositions of the present invention may be subject to various modifications and variations without departing from the spirit or scope of the invention. Thus, the present invention is intended to cover modifications and variations thereof provided they come within the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually incorporated by reference.

Claims (11)

1. An antibody or antigen-binding fragment thereof having binding specificity for wild-type human signal regulatory protein alpha (SIRPa), wherein said antibody or fragment thereof comprises a heavy chain variable region (VH) comprising the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, having the amino acid sequences VH CDRH1, VH CDRH2 and VH CDRH3 SEQ ID: 43, respectively, and a light chain variable region (VL) containing the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, having the amino acid sequences VL CDRL1, VL CDRL2 and VL CDRL3 SEQ ID NO: 45 respectively. 2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that VH CDR1, VH CDR2 and VH CDR3 SEQ ID NO: 43 and VL CDR1, VL CDR2 and VL CDR3 SEQ ID NO: 45 are defined according to the Kabat numbering system. 3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that VH CDR1, VH CDR2 and VH CDR3 SEQ ID NO: 43 and VL CDR1, VL CDR2 and VL CDR3 SEQ ID NO: 45 are defined according to the Chothia numbering system. 4. The antibody or antigen-binding fragment thereof according to claim 1, wherein said heavy chain variable region has the amino acid sequence of SEQ ID NO: 43, and said light chain variable region has the amino acid sequence of SEQ ID NO: 45. 5. A composition for the treatment of cancer containing an antibody or fragment thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier. 6. The composition according to claim 5, additionally containing a second antibody having specificity for the tumor antigen. 7. The composition according to claim 6, characterized in that said second antibody is a tumor opsonizing antibody. 8. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4 for the treatment of cancer. 9. Use according to claim 8, characterized in that said cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer and thyroid cancer. 10. The use of claim 8, further comprising administering to said patient a second antibody targeting a protein that triggers tumor opsonization. 11. Use according to claim 10, characterized in that said protein is selected from the group consisting of CD19, CD20, EGFR, HER2, CD3, CD16, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, VEGFR, TIGIT, claudin 18.2, CD24, GPC3, II13RA2, 4-1BB, CCR8 and SMET.