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WO2025171401A2 - Antibodies against cd99 and methods of use thereof - Google Patents

Antibodies against cd99 and methods of use thereof

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Publication number
WO2025171401A2
WO2025171401A2 PCT/US2025/015295 US2025015295W WO2025171401A2 WO 2025171401 A2 WO2025171401 A2 WO 2025171401A2 US 2025015295 W US2025015295 W US 2025015295W WO 2025171401 A2 WO2025171401 A2 WO 2025171401A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
amino acid
cells
light chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/015295
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French (fr)
Other versions
WO2025171401A3 (en
Inventor
Wayne A. Marasco
Allison F. O'NEILL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dana Farber Cancer Institute Inc
Original Assignee
Dana Farber Cancer Institute Inc
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Application filed by Dana Farber Cancer Institute Inc filed Critical Dana Farber Cancer Institute Inc
Publication of WO2025171401A2 publication Critical patent/WO2025171401A2/en
Publication of WO2025171401A3 publication Critical patent/WO2025171401A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • aspects of the invention are directed anti-CD99 antibody compositions.
  • the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, light chain, or a combination thereof.
  • the light chain comprises CDR1 comprising SDINVGSYN (SEQ ID NO: 23), KLGNTY (SEQ ID NO: 20), ALPKQY (SEQ ID NO: 26), SSDVGGYNY (SEQ ID NO: 55), SGSIATN-(X 15 ) (SEQ ID NO: 58), SLRSYY (SEQ ID NO: 63), or SSNIGSNY (SEQ ID NO: 66); CDR2 comprising YYSDSNK (SEQ ID NO: 24), QD(X4) (SEQ ID NO: 28), DVS (SEQ ID NO: 56), KDS (SEQ ID NO: 98), EDN (SEQ ID NO: 59), KDD (SEQ ID NO: 61), GKN (SEQ ID NO: 64), RNN (SEQ ID NO: 67); and/or CDR3 comprising MIWHSSAWV (SEQ ID NO: 25), QAWDSSTAV (SEQ ID NO: 22), FSYAGDNRGV (SEQ ID NO: 25), Q
  • X1, X2, X3, or X4 is a hydrophilic polar amino acid residue.
  • the hydrophilic polar amino acid residue is Threonine (T), Asparagine (N), Lysine (K), Histidine (H), or Serine (S).
  • X1 is a neutral non-polar amino acid residue, for example the amino acid residue is Proline (P) or Tyrosine (Y).
  • X 1 X 2 X 3 is STH, PSH, TNY, or SKY.
  • X4 is S.
  • X4 is N.
  • X 9 or X 10 is a hydrophilic polar amino acid residue, for example the amino acid residue is Asparagine (N), Serine (S), Threonine (T), Arginine (R).
  • X11 is a neutral non-polar amino acid residue, for example the amino acid residue is Phenylalanine (F) or Tyrosine (Y).
  • X9X10X11 is RNY, NTF, or SSY.
  • X5, X6, X 7, or X 8 is a hydrophilic polar amino acid residue, for example the amino acid residue is Asparagine (N), Serine (S), Lysine (K), Histidine (H), Arginine (R), or Glutamate (E).
  • X5 or X8 is a non-polar amino acid residue, for example the amino acid residue is Phenylalanine (F) or Leucine (L).
  • X 5 is Serine (S) or Phenylalanine (F).
  • X6X7X8 is HKE, SNK, or SRL.
  • X12 or X13 is a hydrophilic polar amino acid residue, for example the amino acid residue is Serine (S) or Arginine (R).
  • X13 or X14 is a hydrophobic, non-polar amino acid residue, for example the amino acid residue is phenylalanine (F) or glycine (G).
  • X 12 X 13 X 14 is RGF or SRG.
  • X15 is a non-polar amino acid residue, for example X15 phenylalanine (F), or tyrosine (Y).
  • the antibody is fully human.
  • An aspect of the invention is directed to an antibody composition comprising at least one antibody, wherein the at least one antibody comprises two heavy chains and two light chains.
  • the heavy chain CDRs are identical to reference germline CDRs found between residues 26 and 33, residues 51 and 58, and residues 97 and 113 according to IMGT numbering of SEQ ID NO: 29, or between residues 97 and 108 according to IMGT numbering of SEQ ID NO: 33, or between residues 97 and 110 according to IMGT numbering of SEQ ID NO: 34, except that at least one of the heavy chain CDRs differs by a single amino acid substitution relative to its reference CDR.
  • the light chain CDRs are identical to reference germline CDRs found between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:30, or between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:110 or between residues 26 and 34, residues 52 and 58, and residues 97 and 104 according to IMGT numbering of SEQ ID NO: 31, except that at least one of the light chain CDRs differs by a single amino acid substitution relative to its reference CDR.
  • the antibody composition binds to an epitope that comprises amino residues at positions 23 to 123 of the extracellular domain of CD99 comprising SEQ ID NO: 32.
  • the antibody is fully human.
  • the antibody is a monoclonal antibody.
  • the antibody is an IgG.
  • the antibody is monospecific or bispecific.
  • the antibody has a binding affinity of at least 1.0 x10 -9 M.
  • the heavy chain CDRs include one or more of the following residue substitutions: 30Pro or 30 or 31Ser; 31Asn or 31or 32Lys; or 32His relative to SEQ ID NO: 29 or SEQ ID NO: 34; or 103Gly relative to SEQ ID NO: 33.
  • the light chain CDRs include one or more of the following residue substitutions: 26Ala; 28Pro; 29Asn or 29Lys; 30Thr or 30Gln; 51Asn relative to SEQ ID NO: 30; or 57Asn; 100His; 102Ser; or 104Trp relative to SEQ ID NO: 31.
  • An aspect of the invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising: one or more antibody compositions comprising at least one antibody, wherein the at least one antibody comprises two heavy chains and two light chains.
  • the light chain CDRs are identical to reference germline CDRs found between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:30, or between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:110 or between residues 26 and 34, residues 52 and 58, and residues 97 and 104 according to IMGT numbering of SEQ ID NO: 31, except that at least one of the light chain CDRs differs by a single amino acid substitution relative to its reference CDR.
  • the antibody composition binds to an epitope that comprises amino residues at positions 23 to 123 of the extracellular domain of CD99 comprising SEQ ID NO: 32.
  • the heavy chain CDRs include one or more of the following residue substitutions: 30Pro, 32His of SEQ ID NO: 29 relative to SEQ ID NO: 40.
  • the heavy chain CDRs include one or more of the following residue substitutions: 31Asn, 103Gly of SEQ ID NO: 33 relative to SEQ ID NO: 40.
  • the heavy chain CDRs include one or more of the following residue substitutions: 30 or 31Ser; 31or 32Lys of SEQ ID NO: 34 relative to SEQ ID NO: 40.
  • the light chain CDRs include one or more of the following residue substitutions: 29Asn; 30Thr; or 51Asn of SEQ ID NO: 30 relative to SEQ ID NO: 41. In embodiments, the light chain CDRs include one or more of the following residue substitutions: 26Ala; 28Pro; 29Lys; or 30Gln of SEQ ID NO: 110 relative to SEQ ID NO: 41. In embodiments, the light chain CDRs include one or more of the following residue substitutions: 57Asn; 100His; 102Ser; or 104Trp of SEQ ID NO: 31 relative to SEQ ID NO: 42. In embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine.
  • An aspect of the invention is directed to a cell producing an antibody described herein.
  • An aspect of the invention is directed to a genetically engineered cell, wherein the genetically engineered cell expresses and/or secretes an antibody described herein.
  • An aspect of the invention is directed to methods of treating cancer.
  • the method comprises administering to a subject in need thereof a composition comprising an antibody described herein or pharmaceutical composition described herein.
  • the cancer expresses CD99.
  • the cancer comprises Ewing Sarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, astrocytoma, glioblastoma, pancreatic endocrine tumor, GI/pulmonary neuroendocrine tumor, prostate cancer, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, lymphoma, or a combination of the CD-99 expressing cancers described herein.
  • An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:3, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:4.
  • An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:7, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:8.
  • An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:11, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:12.
  • An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:71, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:72.
  • An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:75, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:76.
  • An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:79, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:80.
  • An aspect of the invention is directed to a nucleic acid encoding an antibody described herein.
  • the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 5 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 6; the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 1 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 2; or the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 9 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 10.
  • An aspect of the invention is directed to a cell comprising the vector comprising the nucleic acid encoding an antibody described herein.
  • An aspect of the invention is directed to a kit comprising at least one antibody composition described herein; a syringe, needle, or applicator for administration of the at least one antibody or fragment thereof to a subject; and instructions for use.
  • An aspect of the invention is directed to a probe comprising an antibody described herein, and wherein the antibody is further coupled to an imaging agent.
  • An aspect of the invention is directed to an isolated anti-CD99 antibody comprising an antibody described herein, wherein the antibody competes with binding to CD99 or binds the same epitope as PILR ⁇ or an anti-PILR ⁇ antibody.
  • An aspect of the invention is directed to a chimeric antigen receptor (CAR) cell that expresses and secretes an antibody described herein.
  • the antibody comprises an anti-CD99 antibody.
  • FIG.1 shows the scFv binding affinity by ELISA as a function of soluble CD99 concentration (range: 0.3125 ⁇ g/mL to 2 ⁇ g/mL).
  • FIG.2 shows FACs to assess anti-CD99 mAb binding of clone 2, at varying concentrations (range: 0.008 ⁇ g/mL to 2 ⁇ g/mL), to A673 Ewing sarcoma cells. The red histogram represents cells alone.
  • FIG.3 panels A-C, demonstrate cell death of three human-derived Ewing sarcoma cell lines (CADO-ES1, TC32, A673) following incubation with anti-CD99 mAb clones and rabbit serum complement.
  • Panels D-F demonstrate Ewing cell death following incubation with anti-CD99 mAb and Jurkat effector cells.
  • FIG.4 shows representative markers, corresponding to tumor infiltrating immune cells, to be assessed by IHC and flow cytometry.
  • FIG.5 shows a timeline of events leading to the creation of a humanized Ewing xenograft model.
  • FIG.6 shows A) FACs demonstrating A673 Ewing sarcoma cells alone (red), A673 cells incubated with a non-specific FITC-bound isotype control (blue), A673 cells incubated with 10 ⁇ g/mL of NOA1 (orange), 2 ⁇ g/mL of NOA2 (light green), and 2 ⁇ g/mL of NOA3 (dark green). Three samples were incubated with a mouse anti-human FITC-conjugated secondary.
  • Panel B shows FACs demonstrating Kelly neuroblastoma alone and in each of the conditions described herein.
  • FIG.7 shows OctetRED analysis for NOA2 (Panel A, NOA2 F(ab’)2 concentrations of 15nM (Sensor A), 13.5nM (Sensor B), 12.5nM (Sensor C), 11.4nM (Sensor D), 10nM (Sensor E), and 0 nM (Sensor F)) and for NOA3 (Panel B, NOA3 F(ab’)2 concentrations of 25nM (Sensor A), 22.5nM (Sensor B), 20nM (Sensor C), 17.5nM (Sensor D), 15nM (Sensor E), 12.5nM (Sensor F), 10
  • FIG.8 shows panels A-C, which demonstrate cell death of three human-derived Ewing sarcoma cell lines (CADO-ES1, TC32, A673) by ADCC following incubation with anti-CD99 mAb and Jurkat effector cells.
  • Panels D-F demonstrate Ewing cell death by CDC following incubation with anti-CD99 mAb clones and rabbit serum complement.
  • FIG.9 provides confocal microscopy images demonstrating NOA2 internalization.
  • Panels A and F demonstrate light-field images of a singular TC32 cell
  • Panels B and G Hoescht nuclear staining
  • Panels C and H DiD dye in a membranous distribution with pockets of dye in the cytoplasm
  • Panels D and I FITC-bound NOA2 in a membranous distribution also with pockets of dye in the cytoplasm
  • Panels E and J co-localization of DiD and FITC-bound NOA2 throughout the cytoplasm indicating endocytosis-driven NOA2- propagated CD99 internalization.
  • FIG.10 shows mean fluorescence measured by BLI for mice treated with an isotype control versus NOA2.
  • Panel A demonstrates tumor growth arrest in mice treated with NOA2 antibody 24 hours following PBMC infusion.
  • Panel B displays tumors from mice treated with IgG or NOA2 embedded in paraffin and stained with hematoxylin eosin, anti-CD99, and human anti-CD45, mouse anti-CD14, anti-IGF1R and Ras, and TUNEL to assess for apoptosis.
  • FIG.11 shows panel A, which provides light microscopy images of macrophages actively engulfing Ewing sarcoma cells following incubation with NOA2 (labeled red with DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 PKH26).
  • Panel B demonstrates a graphical representation of PKH26+/CD14+ cell count (i.e. Ewing cells engulfed by macrophages) following treatment with NOA2.
  • FIG.12 shows panel A, which demonstrates ELISA results of PILR ⁇ :CD99 binding in the presence of varying concentrations of PILR ⁇ .
  • Panel B shows PILR ⁇ binding to A673 Ewing cells in the presence of different concentrations of anti-CD99 antibody.
  • Panel C illustrates an interaction between macrophage PILR ⁇ and Ewing cell CD99 and the role of anti-PILR ⁇ or anti-CD99 antibodies in the disruption of the binding.
  • NOA3 heavy chain amino acid sequence that follows the NOA2 sequence in the alignment is SEQ ID NO: 34.
  • Light chain germline IGLV3-1*01 amino acid sequence is SEQ ID NO: 41.
  • NOA1 light chain amino acid sequence that follows the germline sequence in the alignment is SEQ ID NO: 30.
  • NOA3 light chain amino acid sequence that follows the NOA1 sequence in the alignment is SEQ ID NO: 110.
  • Light chain germline IGLV5-37*01 amino acid sequence is SEQ ID NO: 42.
  • NOA2 light chain amino acid sequence that follows the germline sequence in the alignment is SEQ ID NO: 31.
  • FIG.15 depicts FACS graphs as well as EC50 graphs.
  • Panel A depicts FACs for A673 Ewing sarcoma cells alone (purple), A673 cells incubated with a non-specific DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 FITC-bound isotype control (orange), A673 cells incubated with 10 ⁇ g/mL of NOA1 (blue), 2 ⁇ g/mL of NOA2 (red), and 2 ⁇ g/mL of NOA3 (green). Three samples were incubated with a mouse anti-human FITC-conjugated secondary. The lower panel depicts FACs for Kelly neuroblastoma alone and in each of the conditions described herein.
  • Panel B depicts FACs demonstrating diminishing fluorescence with decreasing concentrations of NOA2 bound to A673, TC32, CADO-ES1, TC71 and TTC466 Ewing sarcoma cells.
  • Panel C displays the EC50 generated from the geometric mean fluorescence to account for relative CD99 expression per cell line.
  • FIG.16 shows graphs and light microscopy micrographs. Panels A-C demonstrate cell death of three human-derived Ewing sarcoma cell lines (CADO-ES1, TC32, A673) by ADCC following incubation with anti-CD99 mAb and Jurkat effector cells.
  • CADO-ES1, TC32, A673 human-derived Ewing sarcoma cell lines
  • Panel D shows light images obtained utilizing Celigo demonstrating aggregated, plump macrophages actively engulfing Ewing sarcoma cells following incubation with NOA2 (labeled red with PKH26), i.e. ADCP. This finding was more prominent for the p53 wildtype cell lines CADO-ES1 and TC32 (Panel A, arrows).
  • Panel E demonstrates a dose-independent increase in PKH26+/CD14+ cells (i.e. Ewing cells engulfed by macrophages) following treatment with NOA2 in CADO-ES1 and TC32.
  • FIG.17 shows mean fluorescence graphs as well as light microscopy images.
  • Panel B displays tumors from mice treated with IgG or NOA2 embedded in paraffin and stained with hematoxylin eosin, anti-CD99, human anti-CD45/CD33/CD16, mouse anti-CD14, anti-IGF- 1R, IGF-1, Ras, and TUNEL to assess for apoptosis.
  • FIG.18 shows graphs of chemotaxis experiments.
  • Panel A indicates chemotaxis of human monocytes in a dose-dependent fashion through a transwell membrane towards IGF-1 (with MCP-1 included as a positive control).
  • FIG.19 shows a graph of flow cytometry of the immune cell compartment of subcutaneous Ewing tumors grown in humanized mice demonstrate a robust CD33+ infiltrate. Grouping tumors from more than one NOA2 treatment timepoint (Panel A), allows a calculation of statistical significance between groups.
  • FIG.20 shows graphs of binding assays. Human PILR ⁇ binds to both plate-bound and Ewing cell-surface human CD99. Panel A demonstrates ELISA results confirming PILR ⁇ -CD99 binding in a PILR ⁇ -dose dependent fashion. Panel B shows that with escalating concentrations of anti-CD99 antibody, there is diminished PILR ⁇ binding to A673 Ewing cells.
  • FIG.21 shows graphs of cell surface expression.
  • Panel A demonstrates cell surface expression of CD47 in CADO-ES1, TC32, and A673 following pre-treatment with NOA2. While three cell lines demonstrate an increase in CD47 expression following pre-treatment with varying doses of NOA2, A673 demonstrates the largest increase in expression at the highest concentrations of NOA2.
  • FIG.22 shows a schematic of the downstream effects of NOA2 binding to membranous CD99 on Ewing sarcoma cells resulting in increased IGF-1 transcription and DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 secretion. IGF-1 then acts to further upregulate the IGF-1/IGF-1R pathway through an autocrine loop.
  • FIG.23 shows sensorgrams of antibody binding. OctetRED analysis for NOA2 and 3.
  • Panel A NOA2 F(ab’)2 concentrations utilized were as follows: 15nM (Sensor A), 13.5nM (Sensor B), 12.5nM (Sensor C), 11.4nM (Sensor D), 10nM (Sensor E), and 0 nM (Sensor F).
  • Panel B NOA3 F(ab’)2 concentrations utilized were as follows: 25nM (Sensor A), 22.5nM (Sensor B), 20nM (Sensor C), 17.5nM (Sensor D), 15nM (Sensor E), 12.5nM (Sensor F), 10nM (Sensor G), and 0nM (Sensor H).
  • Panel B shows FACS demonstrating diminishing fluorescence with decreasing concentrations of NOA2 bound to A673, TC32, CADO ⁇ ES1, TC71 and TTC466 Ewing sarcoma cells.
  • Panel C shows OctetRED analysis for NOA2.
  • NOA2 F(ab’)2 concentrations utilized were as follows: 15nM (Sensor A), 13.5nM (Sensor B), 12.5nM (Sensor C), 11.4nM (Sensor D), 10nM (Sensor E), and 0 nM (Sensor F).
  • Panel D provides the heavy and light chain sequences, comparative to germline, for NOA2.
  • FIG.25 Panels A ⁇ C show cell death of three human ⁇ derived Ewing sarcoma cell lines (CADO ⁇ ES1, TC32, A673) by ADCC following incubation with anti ⁇ CD99 mAb and Jurkat effector cells. Kelly neuroblastoma cells were used as a negative control.
  • Panel D shows light images obtained utilizing Celigo demonstrating aggregated, plump macrophages actively engulfing Ewing sarcoma cells following incubation with NOA2 (labeled red with PKH26), DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 i.e. ADCP.
  • Panel B shows tumors from mice treated with IgG or NOA2 embedded in paraffin and stained by immunohistochemistry.
  • Tumors from mice treated with NOA2 demonstrate a more prominent human CD45+ infiltrate, human CD16+/CD33+ staining myeloid cells, and plump, activated ⁇ appearing mouse CD14+ cells compared with tumors from mice treated with IgG.
  • NOA2 ⁇ treated tumors also stain for TUNEL indicating apoptosis.
  • Panel C shows the numbers of each infiltrating immune cell type counted in three 10x high ⁇ powered fields.
  • Panel D shows flow cytometry results of the immune cell infiltrate isolated from subcutaneous tumors in a follow ⁇ up humanized mice experiment. Tumors were isolated from one mouse in each treatment cohort at each of the timepoints indicated.
  • FIG.27 Panel A shows immunohistochemical staining of tumors from the micro ⁇ metastatic mouse model depicted in Figure 3A ⁇ C. Tumors from mice treated with NOA2, as compared with mice treated with IgG, show upregulation of IGF ⁇ 1, Ras, and IGF ⁇ 1 implicating the IGF ⁇ 1/Ras ⁇ Rac ⁇ 1 pathway downstream of CD99 binding. Panel B shows results to a chemotaxis assay investigating whether IGF ⁇ 1 is responsible for monocyte migration. Human monocytes migrate through a transwell membrane towards concentrations of IGF ⁇ 1 greater than 10 ng/mL.
  • FIG.29 provides heavy and light chain sequences and comparative human germlines for the top three binding anti-CD99 scFv’s (NOA1, 2, and 3). Red letters indicate single nucelotide mutations (SNMs).
  • FIG.30 shows repeat FACS analysis to depict the geometric mean fluorescence of A673, TC71 and TTC466.
  • FIG.31 shows CD47 in CADO-ES1, TC32, and A673 following pre-treatment with NOA2.
  • the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity when compared a specified region or the full length of any one of the anti-CD99 antibodies described herein.
  • the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 higher nucleic acid identity when compared to a specified region or the full length of any one of the nucleic acid sequences encoding anti-CD99 antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and/or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology.
  • peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids can refer to “polypeptide” herein, and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
  • Polypeptide can also refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
  • amino acid sequences one of skill in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence is collectively referred to herein as a "conservatively modified variant".
  • the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • amino acid sequences one of skill in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence is collectively referred to herein as a "conservatively modified variant".
  • the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, 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), nonpolar 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 (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • an “antibody” or “antigen-binding polypeptide” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen.
  • An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof.
  • antibody can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen.
  • Non-limiting examples a complementarity determining region (CDR) of a heavy or light chain 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 portion of a binding protein.
  • CDR complementarity determining region
  • the term “antibody” can refer to an immunoglobulin molecule and immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin immunoglobulin
  • immunologically active portions of an immunoglobulin (Ig) molecule i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Ig immunoglobulin
  • Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, antigen-binding fragments (Fab), Fab' and F(ab')2 fragments, scFvs, and Fabexpression libraries.
  • antibody fragment or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab′)2, F(ab)2, Fab′, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 antibody.
  • antibody fragment can include aptamers (such as spiegelmers), minibodies, and diabodies.
  • antibody fragment can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanizedor chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, dAb (domain antibody), minibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.
  • polyclonal, monoclonal, multispecific, human, humanizedor chimeric antibodies single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain
  • a “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins.
  • a single chain Fv (“scFv”) polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide- encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883).
  • the regions are connected with a short linker peptide of ten to about 25 amino acids.
  • the linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.
  • This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
  • a number of methods have been described to discern chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule, which will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S.
  • the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated by hybridomas, B cells, or genetically engineered host cells.
  • the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • the antigen-binding surface is DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs.”
  • CDRs complementarity-determining regions
  • VH and VL regions, which contain the CDRs, as well as frameworks (FRs) of the CD99 antibodies are shown in Tables 1A-10B.
  • the nucleic acid and amino acid sequence of the monoclonal human anti-CD99 antibodies are provided herein: Table 1A.
  • VH chain of Ab NOA10 (IGHV3-33*03, IGHD3-10*01, IGHJ6*03) tt a ct cc t a gg Table 10B.
  • Ab NOA10 Variable Region amino acid sequences VH chain of Ab NOA10 (IGHV3-33*03 IGHD3-10*01 IGHJ6*03) V ) S [0080] Table 11. Amino Acid Sequences of Heavy and Light Chain CDRs.
  • amino acids comprising the CDRs and the framework regions, respectively can be readily identified for a heavy or light chain variable region by one of ordinary skill in the art, since they have been previously defined (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)). [0082] Where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include such meanings unless explicitly stated to the contrary.
  • CDR complementarity determining region
  • Kabat numbering refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). [0084] In addition to table herein, the Kabat number system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue.
  • CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue.
  • CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR- H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid.
  • CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue.
  • CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues.
  • X 1 can be a neutral non-polar amino acid residue (e.g., proline (P) Tyrosine (Y)).
  • V L CDR2 can be QD-X 4 (SEQ ID NO: 28).
  • X4 can be a hydrophilic polar amino acid residue (e.g., Asparagine (N), or Serine (S)).
  • V H CDR2 can be I-X 5 -YDG-X 6 X 7 X 8 (SEQ ID NO: 52).
  • X5, X6, X7, or X8 can be Asparagine (N), Serine (S), Lysine (K), Histidine (H), Arginine (R), Glutamate (E)).
  • X5 or X8 can be a non-polar amino acid residue (e.g., phenylalanine (F), Leucine (L)).
  • VH CDR3 can be AR- X12X13X14-MDV (SEQ ID NO: 53).
  • X 12 or X 13 can be a hydrophilic polar amino acid residue (e.g., Serine (S), Arginine (R)).
  • X13 or X14 can be a hydrophobic, non-polar amino acid residue (e.g., phenylalanine (F), glycine (G)).
  • VH CDR1 can be GFTF- X9X10X11-G (SEQ ID NO: 54).
  • X 9 or X 10 can be a hydrophilic polar amino acid residue (e.g., Asparagine (N), Serine (S), Threonine (T), Arginine (R)).
  • X11 can be a non-polar amino acid residue (e.g., phenylalanine (F), or tyrosine (Y)).
  • V L CDR1 can be SGSIATN- X 15 (SEQ ID NO: 58).
  • X15 can be a non-polar amino acid residue (e.g., phenylalanine (F), or tyrosine (Y)).
  • hydrophobic, non-polar amini residues include glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and tryptophan (W).
  • Hydrophilic polar amino acid residues can include serine (S), arginine (R), threonine (T), cysteine (C), asparagine (N), glutamine (Q), lysine (K), histidine (H), aspartic acid (D), glutamic acid/glutamate (E), and tyrosine (Y).
  • neutral amino acid residues include serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), tyrosine (Y), glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), arginine (R), and tryptophan (W).
  • tyrosine (Y), tryptophan (W), lysine (K), and methionine (M) can be polar or nonpolar.
  • epitope can include any protein determinant capable of specific binding to an immunoglobulin, a scFv, or a T-cell receptor.
  • the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens.
  • the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three- dimensional antigen-binding site. This quaternary antibody structure forms the antigen- binding site at the end of each arm of the Y.
  • Epitopic determinants can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • antibodies can be raised against N- terminal or C-terminal peptides of a polypeptide.
  • the antibodies are directed to the EC domain of CD99. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3).
  • the antibodies can be directed to DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [0093] CD99 (SEQ ID NO: 32; GenBank Accession No.: CAG29282, 185 amino acids in length): 1 margaalall lfgllgvlva apDGGFDLSD ALPDNENKKP TAIPKKPSAG DDFDLGDAVV 61 DGENDDPRPP NPPKPMPNPN PNHPSSSGSF SDADLADGVS GGEGKGGSDG GGSHRKEGEE 121 ADAPGVIPGI VGAVVVAVAG AISSFIAYQK KKLCFKENAE QGEVDMESHR NANAEPAVQR 181 TLLEK [0094] The lower-case residues correspond to the signal sequence (SS); bolded residues correspond to the extracellular domain (EC); underlined residues correspond to the transmembrane domain (TD); and italicized residues correspond to the cytoplasmic domain
  • immunological binding can refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength, or affinity of immunological binding interactions can be expressed in terms of the equilibrium binding constant (K D ) of the interaction, wherein a smaller K D represents a greater affinity.
  • Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen- binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
  • both the "on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)).
  • the ratio of Koff /Kon enables the cancellation of parameters not related to affinity and is equal to the equilibrium binding constant (K D ). (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473).
  • An antibody of the invention can specifically bind to a CD99 epitope when the equilibrium binding constant (K D ) is ⁇ 10 ⁇ , ⁇ 10 nM, ⁇ 10 pM, or ⁇ 100 pM to about 1 pM, as measured by kinetic assays such as radioligand binding assays or similar assays known to those skilled in the art, such as BIAcore.
  • K D equilibrium binding constant
  • “Specifically binds” or “has specificity to,” can refer to an antibody that binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope.
  • an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it can bind to a random, unrelated epitope.
  • the CD99 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 antibody can be monovalent or bivalent, and comprises a single or double chain. Functionally, the binding affinity of the CD99 antibody is within the range of 10 ⁇ 5 M to 10 ⁇ 12 M.
  • the binding affinity of the CD99 antibody is from 10 ⁇ 6 M to 10 ⁇ 12 M, from 10 ⁇ 7 M to 10 ⁇ 12 M, from 10 ⁇ 8 M to 10 ⁇ 12 M, from 10 ⁇ 9 M to 10 ⁇ 12 M, from 10 ⁇ 5 M to 10 ⁇ 11 M, from 10 ⁇ 6 M to 10 ⁇ 11 M, from 10 ⁇ 7 M to 10 ⁇ 11 M, from 10 ⁇ 8 M to 10 ⁇ 11 M, from 10 ⁇ 9 M to 10 ⁇ 11 M, from 10 ⁇ 10 M to 10 ⁇ 11 M, from 10 ⁇ 5 M to 10 ⁇ 10 M, from 10 ⁇ 6 M to 10 ⁇ 10 M, from 10 ⁇ 7 M to 10 ⁇ 10 M, from 10 ⁇ 8 M to 10 ⁇ 10 M, from 10 ⁇ 9 M to 10 ⁇ 10 M, from 10 ⁇ 5 M to 10 ⁇ 9 M, from 10 ⁇ 6 M to 10 ⁇ 9 M, from 10 ⁇ 7 M to 10 ⁇ 9 M, from 10 ⁇ 8 M to 10 ⁇ 10
  • a CD99 protein of the invention can be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • a CD99 protein or a derivative, fragment, analog, homolog, or ortholog thereof, coupled to a proteoliposome can be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by ascertaining whether the former prevents the latter from binding to CD99.
  • Another way to determine whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to pre-incubate the human monoclonal antibody of the invention with the CD99 protein, with which it is normally reactive, and then add the human monoclonal antibody being tested to determine if the human monoclonal antibody being tested is inhibited in its ability to bind CD99.
  • human monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention.
  • Screening of human monoclonal antibodies of the invention can be also carried out by utilizing CD99 and determining whether the test monoclonal antibody is able to neutralize CD99.
  • ELISA results demonstrate competitive binding between PILR ⁇ and anti-CD99, indicating a shared epitope on CD99. Accordingly, in certain embodiments, the anti-CD99 antibody binds an epitope that overlaps with that of PILR ⁇ and/or anti-CD99.
  • aspects of the invention are drawn to an an-CD99 antibody of the present disclosure, wherein the anti-CD99 antibody competes with binding to CD99 or binds the same epitope as PILR ⁇ or an anti-PILR ⁇ antibody.
  • a first binding protein e.g., antibody or ligand “binds to the same epitope” as a second binding protein (e.g., antibody or ligand) if the first binding protein binds to the same site on a target compound that the second binding protein binds, or binds to a site that overlaps (e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence or other molecular feature (e.g., glycosyl group, phosphate group, or sulfate group)) with the site that the second binding protein binds.
  • overlaps e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence or
  • a first binding protein e.g., antibody or ligand “competes for binding” with a second binding protein (e.g., antibody) if the binding of the first binding protein to its epitope decreases (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) the amount of the second binding protein that binds to its epitope.
  • the competition can be direct (e.g., the first binding protein binds to an epitope that is the same as, or overlaps with, the epitope bound by the second binding protein), or indirect (e.g., the binding of the first binding protein to its epitope causes a steric change in the target compound that decreases the ability of the second binding protein to bind to its epitope).
  • direct e.g., the first binding protein binds to an epitope that is the same as, or overlaps with, the epitope bound by the second binding protein
  • indirect e.g., the binding of the first binding protein to its epitope causes a steric change in the target compound that decreases the ability of the second binding protein to bind to its epitope.
  • Various procedures known within the art can be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof.
  • Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D.
  • the term "monoclonal antibody” or “mAb” or “MAb” or “monoclonal antibody composition”, as used herein, can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.
  • the complementarity determining regions (CDRs) of the monoclonal antibody are identical in the molecules of the population.
  • MAbs contain an antigen binding site capable of immunoreacting with an epitope of the antigen characterized by a unique binding affinity for it.
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
  • the immunizing agent can include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes can be used if cells of human origin are desired, or spleen cells or lymph node cells can be used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59- 103).
  • Immortalized cell lines can be transformed mammalian cells, such as myeloma cells of rodent, bovine and human origin. For example, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Immortalized cell lines that are useful are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • immortalized cell lines can be murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia).
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63)).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • the hybridoma cells can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No.4,816,567 (incorporated herein by reference in its entirety).
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (See U.S. Patent No.4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein. “Humanized antibodies” can be antibodies from non-human species whose light chain and heavy chain protein sequences have been modified to increase their similarity to antibody variants produced in humans.
  • Humanized antibodies are antibody molecules derived from a non-human species antibody that bind the antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen-binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen-binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat.
  • the non-human part of the antibody can bind to the target antigen.
  • a humanized monoclonal antibody can also be referred to a “human monoclonal antibody” herein.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat.
  • “Humanization” is a well-established technique understood by the skilled artisan for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (i.e., rodent) and for improving their activation of the human immune system (See, for example, Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). "Humanization of an anti-CD34 monoclonal antibody DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 by complementarity-determining region grafting based on computer-assisted molecular modeling”.
  • Human monoclonal antibodies such as fully human and humanized antibodies, can be prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al, 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be utilized and can be produced by using human hybridomas (see Cote, et al, 1983.
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S.
  • a non-limiting example of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules.
  • scFv single chain Fv
  • therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced.
  • this technology for producing human antibodies see Lonberg and Huszar Int. Rev. Immunol.73:65-93 (1995).
  • an antibody of interest such as a human antibody, is disclosed in U.S. Patent No.5,916,771.
  • This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • the antibody can be expressed by a vector containing a DNA segment encoding the single chain antibody described herein.
  • Vectors include, but are not limited to, chemical conjugates such as described in WO 93/64701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618), which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g.
  • the vectors can be chromosomal, non-chromosomal or synthetic. Retroviral vectors can also be used, and include moloney murine leukemia viruses. DNA viral vectors can also be used, and include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (See Geller, A. I. et al, J. Neurochem, 64:487 (1995); Lim, F., et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A.
  • HSV herpes simplex I virus
  • Avipox virus vectors result in only a short-term expression of the nucleic acid.
  • Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus (HSV) vectors can be used for introducing the nucleic acid into neural cells.
  • the adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors.
  • the vector chosen will depend upon the target cell and the condition being treated.
  • the introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation.
  • Examples of modes of gene transfer include e.g., naked DNA, CaP0 4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.
  • the vector can be employed to target essentially any target cell.
  • stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a location.
  • the particles can be delivered by intracerebroventricular (icv) infusion using a DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 minipump infusion system, such as a SynchroMed Infusion System.
  • a method based on bulk flow termed convection, has also proven effective at delivering large molecules to extended areas of the brain and can be useful in delivering the vector to the target cell.
  • convection A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and can be useful in delivering the vector to the target cell.
  • Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.
  • These vectors can be used to express large quantities of antibodies that can be used in a variety of ways, for example, to detect the presence of CD99 in a sample.
  • the antibody can also be used to try to bind to and disrupt a CD99 activity.
  • Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No.4,946,778).
  • methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • Antibody fragments that contain the idiotypes to a protein antigen can be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • Heteroconjugate antibodies are also within the scope of the invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies can, for example, target immune system cells to unwanted cells (see U.S.
  • Patent No.4,676,980 and for treatment of HIV infection (see WO 91/00360; WO 92/200373; EP 03089).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.
  • suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.4,676,980.
  • the antibody of the invention can be modified with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC).
  • ADCC antibody- dependent cellular cytotoxicity
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.
  • the antibody e.g., a human antibody or bispecific antibody (bsAb)
  • bsAb bispecific antibody
  • the mAb contains mutations on both chains of the heterodimeric mAb, which completely ablates the ADCC activity.
  • the mutations introduced one or both scFv units of the mAb are LALA mutations in the CH2 domain.
  • the Fc variant comprises reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering).
  • the antibody has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS.
  • the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering).
  • the antibody comprises an IgGl or IgG4 constant region comprising an S228P and a. T299A mutation (EU numbering).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diis
  • bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT
  • a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.
  • MX-DTPA l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid
  • Those of ordinary skill in the art understand that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the invention. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.
  • Coupling can be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In one embodiment, binding is covalent binding. Covalent binding can be achieved by direct condensation of existing side DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 chains or by the incorporation of external bridging molecules.
  • bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the invention, to other molecules.
  • representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines.
  • This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun.133 : 1335- 2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al, Science 238: 1098 (1987)).
  • Non-limiting examples of linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res.44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker.
  • MBS M-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Non-limiting examples of useful linkers that can be used with the antibodies of the invention include: (i) EDC (l-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4- succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2- pyridyldithio) propionamido]hexanoate (Pierce Chem.
  • sulfo- NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates.
  • NHS-ester containing linkers are less soluble than sulfo-NHS esters.
  • the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available.
  • Sulfo-NHS can enhance the stability of carbodimide couplings.
  • Carbodimide couplings when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
  • the antibodies disclosed herein can also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos.4,485,045 and 4,544,545.
  • Non-limiting examples of useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al, J. Biol.
  • bi-specific Antibodies Unlike a monospecific antibody, which recognizes a single antigen or type of antigen, a bi-specific antibody (bsAb) is an antibody comprising two variable domains or scFv units such that the resulting antibody recognizes two different antigens.
  • the present invention provides for bi-specific antibodies that recognize CD99 and a second antigen.
  • An antibody or antigen-binding fragment specific to CD99 can be combined with a second antigen-binding fragment specific to an immune cell to generate a bispecific antibody.
  • the immune cell is selected from the group consisting of a T cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a phagocyte, a natural killer cell, an eosinophil, a basophil, and a mast cell.
  • Molecules on the immune cell which can be targeted include, for example, CD3, CD16, CD19, CD28, and CD64.
  • Exemplary second antigens include tumor associated antigens (e.g., LINGO1, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, Mucins, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, ⁇ V ⁇ 3, ⁇ 5 ⁇ 1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin), cytokines (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF- ⁇ , CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL), and cell surface receptors
  • Bi-specific antibodies of the present invention can be constructed using methods known in the art.
  • the bi-specific antibody is a single polypeptide wherein the two scFv fragments are joined by a long linker polypeptide, of sufficient length to allow intramolecular association between the two scFv units to form an antibody.
  • the bi-specific antibody is more than one polypeptide linked by covalent or non-covalent bonds.
  • bi-specific antibodies of the present invention can be beneficial due to the intrinsic characteristic of IgG4 molecules wherein the Fc region differs from other IgG subtypes in that it interacts poorly with effector systems of the immune response, such as complement and Fc receptors expressed by certain white blood cells.
  • This specific property makes these IgG4-based bi-specific antibodies attractive for therapeutic applications, in which the antibody is required to bind the target(s) and functionally alter the signaling pathways associated with the target(s), however not trigger effector activities.
  • mutations are introduced to the constant regions of the bsAb such that the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb is altered.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • the bi-specific antibodies disclosed herein can be useful in treatment of diseases or medical conditions, for example, cancer.
  • [00152] [00153]
  • Antibodies specifically binding a CD99 protein or a fragment thereof of the invention can be administered for the treatment a CD99 associated disease or disorder.
  • a "CD99-associated disease or disorder” includes disease states and/or symptoms associated with a disease state, where increased levels of CD99 and/or activation of cellular signaling pathways involving CD99 are found.
  • Exemplary CD99-associated diseases or disorders include, but are not limited to, cancer and conditions for which inflammation or auto- immunity are inherent to the pathophysiology.
  • CD99 is frequently overexpressed in many types of tumors, such as pediatric tumors including Ewing sarcoma and specific subtypes of leukemia, as well as synovial sarcoma, malignant peripheral nerve sheath tumors, astrocytoma/glioblastoma, pancreatic endocrine tumors, GI/pulmonary neuroendocrine tumors, prostate cancer, acute lymphoblastic leukemia, acute myeloid leukemia, stem cells in myelodysplastic syndrome, and lymphomas.
  • Antibodies of the invention including bi-specific, polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents.
  • Such agents will be employed to treat or prevent cancer in a subject, increase vaccine efficiency or augment a natural immune response.
  • An antibody preparation for example, one having high specificity and high affinity for its target antigen, is administered to the subject and will have an effect due to its binding with the target. Administration of the antibody can abrogate or inhibit or interfere with an activity of the CD99 protein.
  • a specific dosage and treatment regimen for any patient will depend upon a variety of factors, including the antibodies, variant or derivative thereof used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art.
  • the amount will also depend on the individual patient to be 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 by pharmacological and pharmacokinetic principles well known in the art.
  • Antibodies of the invention specifically binding a CD99 protein or fragment thereof can be administered for the treatment of a cancer in the form of pharmaceutical compositions. Principles and considerations involved in preparing therapeutic pharmaceutical compositions comprising the antibody, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 20th ed. (Alfonso R.
  • a therapeutically effective amount of an antibody of the invention can be the amount needed to achieve a therapeutic objective. As noted herein, this can be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target.
  • the amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.
  • the dosage administered to a subject (e.g., a patient) of the antigen- binding polypeptides described herein can be 0.1 mg/kg to 100 mg/kg of the patient's body weight, between 0.1 mg/kg and 20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of the patient's body weight.
  • Human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
  • the dosage and frequency of administration of antibodies of the disclosure can be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
  • Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention can be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight.
  • Common dosing frequencies can range, for example, from twice daily to once a week.
  • the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed that retain the ability to bind the target protein sequence.
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
  • the formulation can also contain more than DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 one active compound as necessary for the indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine (e.g. IL-15), chemotherapeutic agent, or growth-inhibitory agent.
  • the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Sustained-release preparations can be prepared.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • an antibody according to the invention can be used as an agent for detecting the presence of CD99 (or a protein fragment thereof) in a sample.
  • the antibody can contain a detectable label.
  • Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) can be used.
  • the term "labeled", with regard to the probe or antibody can encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • biological sample can include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph.
  • the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of an analyte mRNA includes Northern hybridizations and in situ hybridizations.
  • in vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • ELISAs enzyme linked immunosorbent assays
  • Western blots Western blots
  • immunoprecipitations immunoprecipitations
  • immunofluorescence immunofluorescence
  • In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. [00165] Procedures for conducting immunoassays are described, for example in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol.42, J. R.
  • analyte protein in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • Antibodies directed against a CD99 protein can be used in methods known within the art relating to the localization and/or quantitation of a CD99 protein (e.g., for use in measuring levels of the CD99 protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies specific to a CD99 protein, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain are utilized as pharmacologically active compounds (referred to herein as "therapeutics").
  • An antibody specific for a CD99 protein of the invention can be used to isolate a CD99 polypeptide by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation.
  • Antibodies directed against a CD99 protein (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 bioluminescent materials, and radioactive materials.
  • Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S, 32 P or 3 H.
  • compositions suitable for administration can comprise the antibody or agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Non-limiting examples of such carriers or diluents include, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. [00170] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, transarterial (e.g., injection), intratumoral (e.g., injection), oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • parenteral e.g., intravenous, intradermal, subcutaneous, transarterial (e.g., injection), intratumoral (e.g., injection), oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents
  • the pH can be DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition is sterile and is fluid to the extent that easy syringeability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions can include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • One exemplary embodiment includes an antibody composition that comprises at least one antibody.
  • the antibody can comprise two heavy chains and two light chains.
  • the heavy chain CDRs are identical to reference germline CDRs found between residues 26 and 33, residues 51 and 58, and residues 97 and 113 according to IMGT numbering of SEQ ID NO: 29. In an alternate embodiments, the heavy chain CDRs are identical to reference germline CDRs found between residues 97 and 108 according to IMGT numbering of SEQ ID NO: 33. In other embodiments, the heavy chain CDRs can be identical to reference germline CDRs found between residues 97 and 110 according to IMGT numbering of SEQ ID NO: 34. In embodiments, at least one of the heavy chain CDRs differs by a single amino acid substitution relative to its reference CDR.
  • the heavy chain CDRs include one or more of the following residue substitutions: 30Pro, 32His of SEQ ID NO: 29 relative to SEQ ID NO: 40.
  • the heavy chain CDRs include one or more of the following residue substitutions: 31Asn, 103Gly of SEQ ID NO: 33 relative to SEQ ID NO: 40.
  • the heavy chain CDRs include one or more of the following residue substitutions: 30 or 31Ser; 31or 32Lys of SEQ ID NO: 34 relative to SEQ ID NO: 40.
  • the light chain CDRs are identical to reference germline CDRs found between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:30.
  • the light chain CDRs can be identical to reference germline CDRs found between residues 26 and 34, residues 52 and 58, and residues 97 and 104 according to IMGT numbering of SEQ ID NO: 110.
  • at least one of the light chain CDRs differs by a single amino acid substitution relative to its reference CDR.
  • the light chain CDRs include one or more of the following residue substitutions: 29Asn; 30Thr; or 51Asn of SEQ ID NO: 30 relative to SEQ ID NO: 41.
  • the light chain CDRs include one or more of the following residue substitutions: 26Ala; 28Pro; 29Lys; or 30Gln of SEQ ID NO: 110 relative to SEQ ID NO: 41.
  • the light chain CDRs include one or more of the following residue DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 substitutions: 57Asn; 100His; 102Ser; or 104Trp of SEQ ID NO: 31 relative to SEQ ID NO: 42.
  • the antibody binds to an epitope that comprises amino residues at positions 23 to 123 of the extracellular domain of CD99 comprising SEQ ID NO: 32.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the pharmaceutical compositions can be incorporated into a kit suitable for therapeutic administration of the pharmaceutical compositions.
  • the kit includes an applicator to administer the pharmaceutical composition.
  • the kit includes a syringe, a needle, or other applicator for administration of at least one antibody fragment (as disclosed herein) to a subject.
  • the kit can further include instructions for use.
  • Methods of Treatment refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer.
  • Beneficial clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can refer to prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • the invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a cancer, or other cell proliferation-related diseases or disorders.
  • Such diseases or disorders include but are not limited to, e.g., those diseases or disorders associated with aberrant expression of CD99.
  • the methods are used to treat, prevent or alleviate a symptom cancer.
  • the methods are used to treat, prevent or alleviate a symptom of a solid tumor such as a sarcoma (e.g., Ewing sarcoma or synovial sarcoma).
  • a sarcoma e.g., Ewing sarcoma or synovial sarcoma
  • Non-limiting examples of other tumors that can be treated by embodiments herein comprise lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer , malignant peripheral nerve sheath tumor, astrocytoma, glioblastoma, pancreatic endocrine tumor, GI/pulmonary neuroendocrine tumor, acute lymphoblastic leukemia, acute myeloid leukemia, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 myelodysplastic syndrome, lymphoma , stomach cancer, or a combination thereof. Additionally, the methods of the invention can be used to treat hematologic cancers such as leukemia and lymphoma.
  • the methods can be used to treat, prevent or alleviate a symptom of a cancer that has metastasized.
  • the invention provides methods for preventing, treating or alleviating a symptom cancer or a cell proliferative disease or disorder in a subject by administering to the subject a monoclonal antibody, scFv antibody or bi- specific antibody of the invention.
  • an anti-CD99 antibody can be administered in therapeutically effective amounts.
  • Subjects at risk for cancer or cell proliferation-related diseases or disorders can include patients who have a family history of cancer or a subject exposed to a known or suspected cancer-causing agent.
  • a prophylactic agent can occur prior to the manifestation of cancer such that the disease is prevented or, alternatively, delayed in its progression.
  • tumor cell growth is inhibited by contacting a cell with an anti- CD99 antibody of the invention.
  • the cell can be any cell that expresses CD99.
  • Also included in the invention are methods of increasing or enhancing an immune response to an antigen.
  • An immune response is increased or enhanced by administering to the subject a monoclonal antibody, scFv antibody, or bi-specific antibody of the invention.
  • the immune response is augmented for example by augmenting antigen specific T effector function.
  • the antigen is a viral (e.g. HIV), bacterial, parasitic or tumor antigen.
  • the immune response is a natural immune response.
  • the invention provides a method of increasing vaccine efficiency by administering to the subject a monoclonal antibody or scFv antibody of the invention and a vaccine. The antibody and the vaccine are administered sequentially or concurrently.
  • the vaccine is a tumor vaccine a bacterial vaccine or a viral vaccine.
  • Chimeric antigen receptor (CAR) T-cell therapies DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025
  • Cellular therapies such as chimeric antigen receptor (CAR) T-cell therapies expressing an anti-CD99 antibody as described herein, are also provided.
  • CAR T-cell therapies redirect a patient’s T-cells to kill tumor cells by the exogenous expression of a CAR on a T-cell, for example.
  • a CAR can be a membrane spanning fusion protein that links the antigen recognition domain of an antibody to the intracellular signaling domains of the T-cell receptor and co-receptor.
  • a suitable cell can be used, for example, that can secrete an anti- CD99 antibody of the present invention (or alternatively engineered to express an anti-CD99 antibody as described herein to be secreted).
  • the anti-CD99 “payloads” to be secreted can be, for example, minibodies, ScFvs, IgG molecules, bispecific fusion molecules, and other antibody fragments as described herein.
  • the cell described herein can then be introduced to a patient in need of a treatment by infusion therapies known to one of skill in the art.
  • the patient can have a CD99-associated disease, such as the cancers described herein.
  • the cell e.g., a T cell
  • the cell can be, for instance, T lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination thereof, without limitation.
  • Exemplary CARs and CAR factories useful in aspects of the invention include those disclosed in, for example, PCT/US2015/067225 and PCT/US2019/022272, each of which are hereby incorporated by reference in their entireties.
  • the CD99 antibodies discussed herein can be used in the construction of multi-specific antibodies or as the payload for a CAR-T cell.
  • aspects of the invention can be drawn to a genetically engineered cell comprising a chimeric antigen receptor, wherein the genetically engineered cell expresses and/or secretes an anti-CD99 antibody as described herein.
  • compositions of the invention as described herein can be administered in combination with a chemotherapeutic agent.
  • Chemotherapeutic agents that can be administered with the compositions described herein include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, e
  • compositions of the invention as described herein can be administered in combination with cytokines.
  • Cytokines that can be administered with the compositions 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- ⁇ .
  • the compositions described herein can be administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
  • the compositions described herein can be administered in combination with other immunotherapeutic agents.
  • Non-limiting examples of immunotherapeutic agents include sizumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensit
  • the invention provides for methods of treating cancer in a patient by administering two antibodies that bind to the same epitope of the CD99 protein or, alternatively, two different epitopes of the CD99 protein.
  • the cancer is treated by administering a first antibody that binds to CD99 and a second antibody that binds to a protein other than DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 CD99.
  • the other protein other than CD99 can include, but is not limited to, LIGO1, IGF-1R, and PILRa.
  • the other protein other than CD99 is a tumor- associated antigen.
  • the invention provides administration of an anti-CD99 antibody alone or with an additional antibody that recognizes another protein other than CD99, with cells that are capable of effecting or augmenting an immune response.
  • these cells can be peripheral blood mononuclear cells (PBMC), or any cell type that is found in PBMC, e.g., cytotoxic T cells, macrophages, and natural killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • cytotoxic T cells e.g., cytotoxic T cells, macrophages, and natural killer (NK) cells.
  • the invention provides administration of an antibody that binds to the CD99 protein and an anti-neoplastic agent, such a small molecule, a growth factor, a cytokine or other therapeutics including biomolecules such as peptides, peptidomimetics, peptoids, polynucleotides, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, and proteases.
  • an anti-neoplastic agent such as a small molecule, a growth factor, a cytokine or other therapeutics including biomolecules such as peptides, peptidomimetics, peptoids, polynucleotides, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, and proteases.
  • small molecules include, but are not limited to, inorganic molecules and small organic molecules.
  • Suitable growth factors or cytokines include an IL- 2, GM-CSF, IL-12, and TNF-alpha, as well as checkpoint inhibition polypeptides known in the art, or macrophage dysfunction polypeptides (such as CSF1R). Small molecule libraries are known in the art. (See, Lam, Anticancer Drug Des., 12: 145, 1997.) Poly nucleotides include RNA, DNA, and non-coding RNAs.
  • polynucleotides include, but are not limited to small interfering (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), piwi- interacting RNA (piRNA), small nucleolar RNA (snoRNA), and long noncoding RNA (lncRNA).
  • siRNA small interfering
  • miRNA micro RNA
  • shRNA short hairpin RNA
  • piRNA piwi- interacting RNA
  • snoRNA small nucleolar RNA
  • lncRNA long noncoding RNA
  • the anti-CD99 antibody of the invention is linked to a detectable moiety, for example, so as to provide a method for detecting a cancer cell in a subject at risk of or suffering from a cancer.
  • the detectable moieties can be conjugated directly to the antibodies or fragments, or indirectly by using, for example, a fluorescent secondary antibody. Direct conjugation can be accomplished by standard chemical coupling of, for example, a fluorophore to the antibody or antibody fragment, or through genetic engineering. Chimeras, or fusion proteins can be constructed which contain an antibody or antibody fragment coupled to a fluorescent DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 or bioluminescent protein.
  • the term "labeled", with regard to the probe or antibody can encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject (such as a biopsy), as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect cells that express CD99 in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of CD99 include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • in vivo techniques for detection of CD99 include introducing into a subject a labeled anti-CD99 antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • targeted conjugates that is, conjugates which contain a targeting moiety— a molecule or feature designed to localize the conjugate within a subject or animal at site or sites
  • localization can refer to a state when an equilibrium between bound, "localized", and unbound, "free” entities within a subject has been essentially achieved. The rate at which such equilibrium is achieved depends upon the route of administration. For example, a conjugate administered by intravenous injection can achieve localization within minutes of injection. On the other hand, a conjugate administered orally can take hours to achieve localization. Alternatively, localization can simply refer to the location of the entity within the subject or animal at selected time periods after the entity is administered. By way of another example, localization is achieved when an moiety becomes distributed following administration.
  • a reasonable estimate of the time to achieve localization can be made by one skilled in the art.
  • the state of localization as a function of time can be followed by imaging the detectable moiety (e.g., a light-emitting conjugate) according to the methods of the invention, such as with a photodetector device.
  • the "photodetector device” used can DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 have a high enough sensitivity to enable the imaging of faint light from within a mammal in a reasonable amount of time, and to use the signal from such a device to construct an image.
  • an image appears as scintillating points of light, each representing a single detected photon.
  • a digital image processor By accumulating these detected photons in a digital image processor over time, an image can be acquired and constructed.
  • photon counting imaging the amplitude of the signal carries no significance. The objective is to simply detect the presence of a signal (photon) and to count the occurrence of the signal with respect to its position over time.
  • At least two types of photodetector devices, described herein, can detect individual photons and generate a signal which can be analyzed by an image processor.
  • Reduced-Noise Photodetection devices achieve sensitivity by reducing the background noise in the photon detector, as opposed to amplifying the photon signal. Noise is reduced primarily by cooling the detector array.
  • the devices include charge coupled device (CCD) cameras referred to as "backthinned", cooled CCD cameras. In the more sensitive instruments, the cooling is achieved using, for example, liquid nitrogen, which brings the temperature of the CCD array to approximately -120°C.
  • “Backthinned” refers to an ultra- thin backplate that reduces the path length that a photon follows to be detected, thereby increasing the quantum efficiency.
  • a sensitive backthinned cryogenic CCD camera is the "TECH 512", a series 200 camera available from Photometries, Ltd. (Tucson, Ariz.).
  • Photon amplification devices amplify photons before they hit the detection screen.
  • This class includes CCD cameras with intensifiers, such as microchannel intensifiers.
  • a microchannel intensifier can contain a metal array of channels perpendicular to and co-extensive with the detection screen of the camera.
  • the microchannel array is placed between the sample, subject, or animal to be imaged, and the camera. Most of the photons entering the channels of the array contact a side of a channel DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 before exiting. A voltage applied across the array results in the release of many electrons from each photon collision.
  • Such image processors can be sold as part of systems which include the sensitive photon-counting cameras described herein, and accordingly, are available from the same sources.
  • the image processors can be connected to a personal computer, such as an IBM-compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif), which can be included as part of a purchased imaging system.
  • a personal computer such as an IBM-compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif)
  • the images can be manipulated by a variety of image processing programs (such as "ADOBE PHOTOSHOP", Adobe Systems, Adobe Systems, Mt. View, Calif.) and printed.
  • the biological sample contains protein molecules from the test subject.
  • One preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • kits for detecting the presence of CD99 or a CD99-expressing cell in a biological sample can comprise: a labeled compound or agent capable of detecting a cancer or tumor cell (e.g., an anti-CD99 scFv or monoclonal antibody) in a biological sample; means for determining the amount of CD99 in the sample; and means for comparing the amount of CD99 in the sample with a standard.
  • the standard is, in some embodiments, a non-cancer cell or cell extract thereof.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect cancer in a sample.
  • EXAMPLES [00220] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
  • EXAMPLE 1 [00221] Human Anti-CD99 Monoclonal Antibodies [00222] The treatment for pediatric solid tumor patients traditionally consists of a combination of chemotherapy, surgery, and radiation therapy. While these modalities are successful in curing patients with localized disease they are associated with substantial acute and long-term toxicities.
  • Ewing sarcoma is a solid tumor arising from the bone and soft tissues affecting approximately 250 children, adolescents, and young adults each year. It is a model tumor for study given nearly universal membranous CD99 expression, disparate outcomes for patients with localized versus metastatic disease, and the unmet need for more effective therapies.
  • Ewing sarcoma One potential target with applicability to sarcoma, prostate cancer, leukemias and lymphomas is CD99, though additional efforts are needed to optimize CD99- targeted treatment strategies.
  • Ewing sarcoma will be investigated as a model system to study CD99 targeting. Ewing Sarcoma is a disease with nearly universal membranous CD99 expression, disparate outcomes for patients with localized versus metastatic disease, and an unmet need for more effective therapies.
  • Previous work demonstrated the ability of a 64Cu-labeled anti-CD99 monoclonal antibody (mAb) to detect micro-metastatic lesions in a Ewing sarcoma mouse model with high sensitivity and specificity.
  • mAb monoclonal antibody
  • Aim 1 To define CD99 mAb activity in an Ewing sarcoma humanized mouse model.
  • a Ewing sarcoma humanized mouse model will allow a unique assessment of the Ewing tumor immune milieu, in vivo anti-CD99 mAb induced tumor cytotoxicity, and on-target off-tumor binding so as to further inform the safety and efficacy of an immunotherapeutic human anti-CD99 mAb.
  • Ewing sarcoma is characterized histologically by small, round blue cells and a diffuse membranous immunohistochemical stain for CD99 (or MIC2), a 32kDa type I membrane glycoprotein.
  • Ewing sarcoma is suited for a targeted approach to therapy given diffuse membranous immunohistochemical staining for CD99 (or MIC2).
  • CD99 is a 32kDa type I membrane glycoprotein highly expressed in 100% of Ewing sarcoma specimens and believed to play a role in the differentiation and malignant phenotype of Ewing cells.
  • 4,15- 19 CD99 is biologically linked to EWS:FLI1, the pathognomonic ETS-family translocation present in up to 85% of Ewing tumors. 20
  • Ectopic expression of the EWS:FLI1 translocation in mesenchymal stem cells can induce Ewing cell morphology and CD99 expression.
  • Ewing sarcoma cases harbor a chimeric fusion between the RNA binding protein EWS and an ETS family transcription factor (i.e., EWS:FLI1) creating a fusion oncogene (t(11;22)(q24;q12)) deemed relevant to tumor pathogenesis.
  • EWS:FLI1 ETS family transcription factor
  • TAMs tumor-associated macrophages
  • a humanized Ewing sarcoma mouse model can be used to better understand the tumor immune milieu, mAb- induced cytotoxicity within the human immune context, and on-target off-tumor binding.
  • CD99 expression is greatest on human leukocytes and pancreatic beta islet cells underscoring the importance of on-target off-tumor study as a means by which to anticipate toxicity. 19
  • results from these experiments will identify an anti- CD99 immunotherapeutic that is more broadly applicable to other CD99-positive tumors (such as additional sarcomas, leukemias, lymphomas, and prostate tumors).
  • Fluorescence activated cell sorting was performed to analyze binding of these scFv’s to patient-derived Ewing sarcoma cell lines and the resultant six binding scFv’s were screened by ELISA against serial dilutions of soluble CD99 to identify the top three clones with reproducible, differential binding at concentrations of 0.5 ⁇ g/mL or less (FIG.1). Sequences for the top three human anti-CD99 scFv heavy and light chains (termed clones 1,2, and 3) were cloned into an IgG1 vector for transfection of 293F cells.
  • FACs Fluorescence activated cell sorting
  • Resultant antibodies were purified by protein A and analyzed for in vitro binding by FACs to 5 patient-derived Ewing sarcoma cell lines. Serial dilutions of each antibody were applied to Ewing cells in a 96-well plate demonstrating dose-dependent binding and retained affinity at low concentrations (FIG.2). OctetRED (Biacore) was utilized to determine the equilibrium dissociation constant (KD) for each clone ( ⁇ 4nM). Cell proliferation and viability studies were performed utilizing CellTiter-Glo and MTT assays. Cell death was not observed in isolation (i.e in the absence of immune effector cells), nor did CD99 internalize following antibody engagement.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Aim 1(a) To examine the Ewing sarcoma tumor immune environment in a humanized Ewing sarcoma xenograft model pre- and post-treatment with human anti-CD99 mAb.
  • In vitro data demonstrates the importance of the immune system in propagating Ewing cell death following human anti-CD99 mAb engagement.
  • Use of a humanized mouse DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 for in vivo experiments will allow study of the tumor immune environment, categorizing immune elements present before and after treatment with human anti-CD99 mAb.
  • mice will undergo whole-body irradiation (100cGy) and subsequent intrahepatic injection of 3x104 CD34+ hematopoietic stem cells (HSCs). After 12 weeks, flow cytometric analyses of the blood will be performed to ensure engraftment defined as >20% peripheral human CD45+ cells. Previous studies have shown successful growth of patient-derived xenografts 2-12 weeks following HSC injection.
  • mice will be injected with Ewing sarcoma patient-derived cells (TC32) 8-10 weeks post-receipt of HSCs, utilizing previously vetted approaches for the development of subcutaneous and orthotopic tumors. More specifically, mice will be injected subcutaneously on the right flank with 1 million Ewing sarcoma cells re-suspended in 100uL of PBS. Tumor size will be measured serially with calipers until tumors reach 100mm3 with an anticipated time to tumor growth of 3-4 weeks.
  • Ewing sarcoma orthotopic models will be pursued by intratibial injection of 50,000 Luc-mCh transduced cells (allowing imaging by bioluminescence, BLI) re-suspended in 50uL (50% matrigel, 50% PBS) with a 3-4 week anticipated time frame to tumor formation.
  • BLI bioluminescence
  • 50uL 50% matrigel, 50% PBS
  • Evaluation of the tumor immune microenvironment A cohort of mice will be euthanized at 14-18 weeks (i.e.2-4 weeks following tumor formation), and the tumor immune microenvironment interrogated by immunohistochemistry (IHC), flow cytometry of single cell suspensions, and Nanostring Technology®.29 IHC and flow cytometric analyses will examine cell surface targets representative of a range of immune cells (FIG.4).
  • RNA will be extracted from tumor tissue and a Cancer Immune Nanostring Technology® panel run to examine 760 gene transcripts relevant to tumor infiltrating immune cells and cytokines/chemokines.
  • nSolverTM software will be used to identify transcripts with significant enrichment.
  • a cohort of mice will be treated with 3mg/kg of antibody administered via tail vein twice weekly for 3-5 weeks (see Aim 1(b)). Mice will be euthanized at staggered time points post-treatment and tumors analyzed herein, allowing a comparison of the immune environment pre- and post-therapy.
  • Aim 1(b) To determine the effect of treatment with human anti-CD99 mAb on tumor cell growth.
  • Ewing cell death in vitro following anti-CD99 mAb engagement relies upon the presence of immune effector cells or serum complement. Use of a humanized mouse model will allow a corresponding study of mAb-induced Ewing cell death in vivo in the context of the human immune environment.
  • In vivo cytotoxicity Humanized Ewing xenograft models with subcutaneous or orthotopic tumors (at 100mm3 or 4 weeks of growth, respectively) will be treated via tail vein with anti-CD99 mAb or an isotype control dosed at 3mg/kg twice weekly. Mice will be followed prospectively until tumor progression (>20% increase in size) or onset of graft- versus-host disease. Tumor burden will be serially assessed using calipers (subcutaneous tumors) and BLI (orthotopic tumors). A fraction of mice will undergo MRI to assess changes in tumor dimension, enhancement, or necrosis.
  • anti-CD99 mAb alone or in combination with immune stimulating agents can serve as a therapeutic if experiments demonstrate slowed tumor growth or robust tumor cell death.
  • Anti-CD99 mAb in combination with conventional chemotherapies can be a therapeutic agent if single-agent mAb treatment fail to impact tumor growth.
  • human mAb drug or radionuclide delivery can ultimately require exploration.
  • orthotopic tumors are difficult to propagate in a humanized Ewing xenograft model, we will prioritize subcutaneous tumors for this arm of the study.
  • Aim 1(c) To investigate on-target off-tumor binding of the human anti-CD99 mAb.
  • Methods Toxicity and preferential tumor binding will be assessed using a step- wise approach.
  • Hematologic toxicity Humanized mice will be created as per Aim 1(a) with HSC engraftment at 12 weeks verified by flow cytometry. Mice will then be injected with anti- CD99 mAb and serially bled for assessment of effect of anti-CD99 Ab binding on total white blood cell count.
  • Pancreatic toxicity Human fetal pancreatic cells at 18-24 weeks of gestation will be digested with collagenase and cultured as islet-like cell clusters (ICCs). 30 The ICCs will be digested into single cells and transplanted under the kidney capsule of 6-week old non- humanized NSG mice.
  • mice After three months, 1-2 representative mice will be euthanized with confirmation of subcapsular pancreatic tissue by IHC. Serum human peptide C levels will be measured as a proxy for insulin secretion. Mice will be injected with anti-CD99 mAb and serially bled to track serum peptide C and glucose levels.
  • Preferential binding Three cohorts of NSG mice will be prepared: 1) human blood-reconstituted, 2) human pancreatic tissue-engrafted, and 3) a combination of the two. Ewing sarcoma subcutaneous flank xenografts, which do not metastasize, will be established in each of the three cohorts.
  • mice will be injected with 64Cu- labeled anti-CD99 and imaged with PET; regions-of-interest will be drawn on marrow, pancreatic, and tumor tissue with standardized-uptake-values (SUVs) compared to assess competitive binding.
  • Terminal radiotracer biodistribution studies will assess blood, pancreatic, tumor, and tissue radioactivity.
  • Serum human peptide C levels will again be drawn in cohorts 2) and 3) to assess for pancreatic toxicity.
  • mice from each category can be treated with non-radionuclide bound antibody to assess for effect of competitive binding on tumor growth. Animal studies will follow Animal Care and Use Committee guidelines.
  • Ewing sarcoma cell lines A673, TTC466, CADO-1, and TC32
  • TC32 Ewing sarcoma cell lines
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Bio-Glo Luciferase Assay was utilized to quantitate cell death. Plates were read on a POLARStar Omega plate reader and relative light units (RLU) analyzed for each condition after subtracting for background.
  • RLU relative light units
  • Complement-dependent-cell (CDC) death assays were performed with pooled baby rabbit complement (Cedarlane). Ewing sarcoma cells were cultured overnight in a 96-well plate at 1x 10 6 cells per well. Serial dilutions of anti-CD99 antibody clones or an isotype control were plated with cells the following morning at concentrations ranging from 0.01 ⁇ g/mL to 10 ⁇ g/mL. Rabbit complement was added at a ratio of 1:8 or 1:16.
  • CD20+ Raji cells were incubated with Rituximab as a control. Cells were incubated for 4 hours at 37oC with 5% CO2. Lysis buffer was added to a control well to calculate 100% cytotoxicity. Cells were centrifuged at 4oC for 4-5 minutes at 250g, supernatant was transferred to a new 96-well plate, assay reagent added and stop solution applied after 30 minutes. Plates were read on a Biorad microplate spectrophotometer at 490nm. % cytotoxicity was calculated using the following formula: ([Experimental – Effector Spontaneous – Target Spontaneous]/[Target Maximum – Target Spontaneous]) x 100.
  • ADCP antibody-dependent cellular phagocytosis assays were performed by first isolating human monocytes from human peripheral blood mononuclear cells (PBMCs) by plating PBMCs in MDM medium (DMEM with 10% heat-inactivated human serum, 1% penicillin/streptomycin, 1% L-glutamine, and 50ng/mL GM-CSF) in a Petri dish for 3 days at 37oC with 5% CO2. At the end of three days, the supernatant was removed and MDM media replenished to further culture the adherent macrophage precursors.
  • MDM medium DMEM with 10% heat-inactivated human serum, 1% penicillin/streptomycin, 1% L-glutamine, and 50ng/mL GM-CSF
  • Engulfed target cells were defined as PKH26+/CD14+.
  • Internalization [00270] 400,000 Ewing sarcoma cells were stained with DiD Lypophilic tracer (ThermoFisher Scientific), plated, and cultured overnight at 37oC with 5% CO2 in phenol- free RPMI on uncoated MatTek coverslip-bottom 35mm dishes. The following day, cells were stained with biotin-avidin FITC-conjugated NOA2 at 2 ⁇ g/mL and incubated for 2 hours, 1 hour and 0.5 hours at 37oC with 5% CO2.
  • the NOA2 heavy chain was of IGHV5-51*03, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 IGHD1-26*01, IGHJ3*02 germline origin and harbored 8 SNMs and the NOA3 heavy chain was of IGHV5-51*01, IGHD3-16*01, IGHJ3*02 germline origin and harbored 7 SNMs.
  • the NOA1 light chain was of IGLV3-1*01, IGLJ1*01 germline origin harboring 9 SNMs
  • the NOA2 light chain was of IGLV5-37*01
  • the NOA3 light chain was of IGLV3-1*01, IGLJ3*02 germline harboring 6 SNMs (FIG.14).
  • Binding of anti-CD99 antibodies to Ewing sarcoma cells by flow cytometry [00275] NOA1, 2 and 3 were subcloned, expressed in 293F cells, and purified as IgG1 antibodies.
  • NOA1 demonstrated limited binding at concentrations as high as 10 ⁇ g/mL whereas NOA2 (light green) and 3 (dark green) demonstrated reproducible binding at concentrations of 2 ⁇ g/mL (FIG.6A). None of the clones bound to CD99-negative Kelly neuroblastoma cells (FIG. 6B). The relative affinity of NOA2 and 3 was determined by flow cytometric saturation binding studies against three independent CD99+ Ewing sarcoma cell lines. (A673 depicted in FIG.6C).
  • EC 50 was determined for each cell line by plotting the geometric mean fluorescence against concentration resulting in an EC50 of 0.125 ⁇ g/mL for NOA2 and 0.1 ⁇ g/mL for NOA3.
  • Kinetic analysis of anti-CD99 antibody binding by OctetRED Analysis was pursued to determine the equilibrium dissociation constants (KD) of NOA2 and 3.
  • the K D for NOA2 was calculated at 3.35 x 10 -9 M +/- 4.60 x 10 -11 M from analysis of F(ab’)2 concentrations between 10 and 15nm with a Kon of 4.15 x 10 5 +/- 4.04 x 10 3 (1/Ms) and a K off of 1.39 x 10 -3 +/- 1.35 x 10 -5 (1/s).
  • the K D for NOA3 was calculated at 1.86 x 10 -9 M +/- 1.65 x 10 -10 M from analysis of F(ab’)2 concentrations ranging between 10 and 25nm with a K on of 3.80 x 10 5 +/- 5.13 x 10 3 (1/Ms) and a K off of 7.06 x 10 -4 +/- 6.18 x 10 -5 (1/s). (FIG.7).
  • Anti-CD99 antibodies mediate killing of CD99+ Ewing sarcoma cells by ADCC and CDC
  • ADCC and CDC performed for three antibody clones demonstrated an increase in Ewing cell death as a function of antibody concentration. There was no Ewing cell death in lines incubated with an isotype control. As shown in FIG.8A-C NOA2 and 3 outperformed NOA1.
  • FIG.8D-F demonstrates cytotoxicity by CDC with three anti-CD99 antibody clones; NOA2 outperformed NOA1 and 3 at concentrations greater than 0.1 ⁇ g/mL. There was no CDC- mediated killing with incubation of Ewing cells with an isotype control.
  • NOA2-mediated CD99 internalization [00281] Confocal microscopy was utilized to evaluate CD99 internalization (FIG.9).
  • Live TC32 Ewing cells were stained with a lipophilic membrane dye, plated, and subsequently incubated with FITC-conjugated NOA2 mAb for 0.5, 1 and 2 hours. Nuclear Hoescht stain was added just prior to live cell confocal imaging. Independent of duration of incubation, FITC-bound CD99 was visualized in the cytoplasm, at multiple imaging planes, co-localizing with Did dye indicating NOA2-propagated endocytosis of membrane-bound CD99. Time- lapse microscopy demonstrated intracellular migration of these dual-fluorescent intracytoplasmic bodies.
  • FIG.10 demonstrates tumor growth arrest, as extrapolated by mean luminescence, in mice treated with NOA2 as compared with an isotype control.
  • NOA2 utilized for these studies on the basis of FACs, OctetRED, and ADCC/CDC data indicating superior binding and Ewing tumor cell kill. Following therapy, mice were euthanized, livers dissected, embedded in paraffin, and sectioned.
  • mice treated with NOA2 Hematoxylin-eosin staining demonstrated pockets of central necrosis positive for apoptotic bodies (TUNEL) in tumors of mice treated with NOA2. This was not observed in mice treated with isotype control. Tumor specimens stained only weakly for human CD45+ cells. However, in contrast to mice treated with an isotype control, tumors from mice treated with NOA2 demonstrated a pronounced mouse myeloid cell infiltrate (mouse CD14+). Tumors from mice treated with NOA2 demonstrated upregulation of IGF- 1R and Ras implying activation of the IGF-1R/Ras/Rac1 pathway.
  • TUNEL apoptotic bodies
  • Anti-CD99 antibodies mediate killing of CD99+ Ewing sarcoma cells by ADCP
  • ADCP studies were performed. Marophages isolated from human PBMCs co-incubated with Ewing sarcoma cells and anti-CD99 antibody demonstrate changes in morphology indicative of activation and tumor cell engulfment for p53 wild-type cell lines CADO-ES1 and TC32 (FIG.11).
  • FACs demonstrates an increase in PKH26+/CD14+ cells, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 representing Ewing cells engulfed by macrophages, following exposure to anti-CD99; this does not appear antibody dose-dependent. While there are morphologic changes in macrophage appearance and engulfment of Ewing sarcoma cells in the presence of isotype IgG, these findings are far more pronounced for CADO-ES1 and TC32 lines in the presence of anti-CD99.
  • FIG.12C illustrates the PILR ⁇ :CD99 axis and the postulated role of anti-CD99 and anti- PILR ⁇ in interrupting macrophage:Ewing sarcoma cell interaction.
  • CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling.
  • Parija, T. et al. Type 1 (11;22)(q24:q12) translocation is common in Ewing's sarcoma/peripheral neuroectodermal tumour in south Indian patients. J Biosci 30, 371-376 (2005). 17.
  • IL6 secreted by Ewing sarcoma tumor microenvironment confers anti- apoptotic and cell-disseminating paracrine responses in Ewing sarcoma cells.
  • NOA2 an anti-CD99 antibody, NOA2 binds Ewing sarcoma cells with high affinity. NOA2 can induce Ewing cell death through engagement of macrophages and antibody-mediated cellular phagocytosis in vitro.
  • NOA1 demonstrated limited binding at concentrations as high as 10 ⁇ g/mL whereas NOA2 (red) and 3 (green) demonstrated strong DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 binding at concentrations of 2 ⁇ g/mL (FIG.15A (upper)). None of the clones bound to CD99-negative Kelly neuroblastoma cells (FIG.15A (lower)). NOA2 was carried forth for further studies given superior binding across Ewing cell lines (results not shown). The relative affinity of NOA2 was determined by flow cytometric saturation binding studies against five independent CD99-positive Ewing sarcoma cell lines (FIG.15B).
  • EC 50 was determined by plotting the geometric mean fluorescence against concentration (FIG.15C).
  • Anti-CD99 antibodies mediate killing of CD99+ Ewing sarcoma cells by ADCC and ADCP. In vitro viability and proliferation assays did not result in direct antibody-mediated Ewing cell death in the absence of immune effector cells. To determine if immune effector cells can play a key role in NOA2 antibody-mediated cytotoxicity, ADCC and ADCP were performed. NOA2 triggered Ewing cell death as a function of antibody concentration in ADCC assays, however the magnitude of cell killing was cell line specific.
  • Ewing cell death as assessed by net luminescence was more pronounced for the p53 wildtype cell lines CADO-ES1 and TC32. Ewing cells retained viability when incubated with an isotype control.
  • macrophages isolated from human PBMCs and co- incubated with Ewing sarcoma cells and NOA2 showed aggregation and engulfment of tumor cells (FIG.16D, arrows). Similar to ADCC, these findings were limited to p53 wildtype cell lines CADO-ES1 and TC32.
  • FACs demonstrated an increase in PKH26+/CD14+ cells, representing Ewing cells engulfed by macrophages following NOA2 incubation; the two NOA2 concentrations tested for ADCP (2 & 5 ⁇ g/ml) did not show an antibody dose- dependent effect (FIG.16E).
  • ADCP was most prominent following incubation with 2 ⁇ g/mL indicating that lower concentrations of NOA2 can be more effective for this immune effector function.
  • FIG.17A demonstrates tumor growth arrest at 14 days post-treatment, as extrapolated by mean luminescence, in mice treated with NOA2 as compared with an isotype control.
  • mice were euthanized following therapy and livers dissected, embedded in paraffin, and sectioned (FIG.17B).
  • the representative images demonstrate a more prominent human CD45, mouse CD14, and human CD33 and CD16 infiltrate.
  • PILR ⁇ is a CD99 ligand, and inhibitory receptor, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 that is preferentially expressed on human myeloid cells but also on natural killer cells, dendritic cells, and other granulocytes.
  • PILR ⁇ has been implicated in and can be restrictive of immune cell recruitment and activation.
  • P27-P30 Data in FIG.20A confirms dose-dependent binding of our synthesized human PILR ⁇ to soluble human CD99 by ELISA.
  • PILR ⁇ and NOA2 was next shown to bind Ewing CD99 competitively by FACs indicating a shared CD99 binding epitope (FIG.20B).
  • FACs results demonstrate two distinct cell populations of PILR ⁇ binding, the second of which is more impacted by anti-CD99 dose-escalation.
  • the next series of experiments were aimed at using NOA2 and anti-PILR ⁇ antibody to interrupt the PILR ⁇ :CD99, macrophage:Ewing sarcoma, axis to assess effects on macrophage activity, using TNF- ⁇ secretion as a proxy.
  • TNF- ⁇ secretion wanes naturally over the course of 48 hours from polarized (M1) macrophages incubated in vitro.
  • Binding affinity for these six scFv’s was assessed by ELISA with titration of soluble CD99- DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 Fc concentrations.
  • the top three clones, NOA1, 2, and 3 demonstrated saturable binding at concentrations of 0.125 ⁇ g/mL or less. These scFvs were carried forth for further studies.
  • the germline Ig gene segment usage and somatic hypermutation (SHM) are shown in FIG.14 and Table XX.
  • the NOA2 heavy chain is most closely aligned with the *03 allele of the IGHV5-51 germline gene, the *01 allele of the IGHD1-26 germline gene, and the *02 allele of the IGHJ3 germline segment.
  • the heavy chain harbors 7 single-nucleotide mutations (SNMs) including in CDR1, however CDR2 is in the germline configuration.
  • SNMs single-nucleotide mutations
  • the light chain utilizes IGLV5-37*01 and IGLJ3*02 germline genes and has 6 SNM.
  • the KD for NOA2 was calculated at 3.35 x 10 -9 M +/- 4.60 x 10 -11 M with K on of 4.15 x 10 5 +/- 4.04 x 10 3 (1/Ms) and K off of 1.39 x 10 -3 +/- 1.35 x 10 -5 (1/s).
  • the KD for NOA3 was calculated at 1.86 x 10 -9 M +/- 1.65 x 10 -10 M with Kon of 3.80 x 10 5 +/- 5.13 x 10 3 (1/Ms) and K off of 7.06 x 10 -4 +/- 6.18 x 10 -5 (1/s). (FIG.23).
  • NOA2 a human monoclonal antibody that targets CD99 can recruit and reactivate components of the innate immune system to direct antibody-mediated Ewing sarcoma death both in vitro and vivo. NOA2 can trigger antibody-mediated tumor death through ADCC (Fc ⁇ R-mediated killing by natural killer cells) and ADCP (phagocytosis by macrophages).
  • ADCC Fc ⁇ R-mediated killing by natural killer cells
  • ADCP phagocytosis by macrophages
  • NOA2 In vivo treatment of Ewing tumor-bearing mice with NOA2 prompts an enhanced infiltrate of morphologically activated mouse CD14+ cells, along with an increase in intratumoral human CD33+ cells and tumor growth arrest and apoptosis. NOA2 is also associated with upregulation of IGF-1, potentially contributing to monocyte/macrophage chemotaxis and recruitment. Combination NOA2 and PILR ⁇ blockade in vitro reverses the inhibitory CD99:PILR ⁇ pathway linking Ewing sarcoma cells and macrophages leading to restoration of TNF- ⁇ secretion.
  • wild type p53 is also relevant for cytotoxicity downstream of Fc ⁇ R-engagement.
  • the differences in ADCP killing were reconciled by examining the overexpression of CD47, recognizing that the CD47/SIRP ⁇ axis controls myeloid cell activation.
  • P31 At baseline, human Ewing-derived cell lines express CD47; a more dramatic upregulation of CD47 on the A673 mutant p53 was noted following treatment with NOA2 potentially inhibiting the success of ADCP (FIG.21).
  • p53 mutation status likewise is of limited translational concern, given that the vast majority of Ewing tumors in humans are p53 wildtype at diagnosis.
  • NOA2 is responsible for recruiting activated, intratumoral macrophages that are contributing to tumor growth arrest via ADCP.
  • Cells staining for human CD16 were also present in higher numbers in NOA2-treated tumors.
  • CD16 is expressed both on a subset of monocytes and NK cells; both CD16-expressing monocytes and NK cells are capable of ADCC, an additional contributor to in vivo tumor apoptosis.
  • P34 The study of tumor-infiltrating lymphocytes has received considerable attention, given the striking successes in use of PD-1 inhibitors for adult malignancies. However, these agents are less likely to be of benefit in Ewing sarcoma given that Ewing tumors are mutationally silent and harbor a paucity of T-cells.
  • P18-P20,P32 Pivoting focus towards manipulation of the myeloid compartment is of therapeutic relevance.
  • NOA2 can serve to recruit activated macrophages to the intratumoral microenvironment
  • tumor-protective macrophages are present.
  • P19,P20 The exploration of the PILR ⁇ :CD99 axis addresses whether dormant macrophages can be re-activated through disruption of PILR ⁇ :CD99 binding.
  • Human PILR ⁇ binds to human CD99 and that NOA2 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 blocks this interaction.
  • P35 Macrophages are known to express both surface PILR ⁇ and CD99; co-incubation with saturating concentrations of anti-PILR ⁇ and CD99 antibodies in this setting leads to binding of both of these receptors on the macrophage cell surface and CD99 on the Ewing cell surface (FIG.20D).
  • FOG.20D Ewing cell surface
  • repression of TNF- ⁇ secretion is observed at timepoints of antibody incubation.
  • Introduction of Ewing CD99+ cells to polarized macrophages introduces a new Ewing:macrophage, CD99: PILR ⁇ , binding axis predicted to contribute to macrophage suppression.
  • CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes. Sci Rep 6, 34310, doi:10.1038/srep34310 (2016). [00345] P35 Sun, Y. et al. Evolutionarily conserved paired immunoglobulin-like receptor alpha (PILRalpha) domain mediates its interaction with diverse sialylated ligands. J Biol Chem 287, 15837-15850, doi:10.1074/jbc.M111.286633 (2012).
  • PILRalpha immunoglobulin-like receptor alpha
  • Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, or refractory settings, we explored the downstream immune implications of targeting CD99.
  • NOA2 human anti ⁇ CD99 antibody
  • Ewing sarcoma is a rare tumor of the bone or soft tissues affecting approximately 250 children, adolescents, and young adults each year in the United States. 1 Patients with chemotherapy ⁇ responsive, localized disease have an excellent 5 ⁇ year overall survival of greater than 75%. 2 Conversely, the presence of metastatic disease confers a very poor prognosis; less than 20% of these patients are cured. 3,4 The mainstay of therapy for Ewing sarcoma consists of systemic chemotherapy, surgery and/or radiotherapy. While effective for the cohort of patients with localized disease, these treatment modalities confer the life ⁇ long risk of organ toxicity, infertility, secondary malignancy, or disfigurement impacting quality of life.
  • Ewing sarcoma is characterized histologically by small, round blue cells and a diffuse membranous immunohistochemical stain for CD99 (or MIC2), a 32kDa type I membrane glycoprotein.
  • Ewing sarcoma cases harbor a chimeric fusion between the RNA binding protein EWS and an ETS family transcription factor (i.e., EWS ⁇ FLI1) creating a fusion oncogene (t(11;22)(q24;q12)) deemed relevant to tumor pathogenesis. 14,15 While the published literature indicates an important link between CD99 and EWS ⁇ FLI1, this relationship is incompletely characterized. 16,17 In addition, the Ewing immune landscape has traditionally been described as a “cold” immune state. However, the presence of CD68+ tumor ⁇ associated macrophages (TAMs) has been linked to poor clinical outcomes.
  • TAMs tumor ⁇ associated macrophages
  • Anti ⁇ CD99 antibody blockade has been shown to induce Ewing cell aggregation and caspase ⁇ independent apoptosis 11 , trigger Ewing cell death in p53 wild ⁇ type patient ⁇ derived cell lines 21 , and more recently incite cell death by a process termed methuosis, a form of non ⁇ apoptotic cell death characterized by displacement of the cytosol by vacuoles derived from macropinosomes, involving the IGF1R/Ras/Rac1 signaling pathway. 22
  • methuosis a form of non ⁇ apoptotic cell death characterized by displacement of the cytosol by vacuoles derived from macropinosomes, involving the IGF1R/Ras/Rac1 signaling pathway. 22
  • methuosis a form of non ⁇ apoptotic cell death characterized by displacement of the cytosol by vacuoles derived from macropinosomes, involving the IGF1R/Ras/Rac
  • NOA2 a high ⁇ affinity human anti ⁇ CD99 antibody, NOA2, capable of binding Ewing sarcoma cells, and subsequently explored the in vitro and in vivo activity of this antibody to engage macrophages, impact tumor growth, and modulate macrophage function.
  • NOA2 can induce Ewing cell death through engagement of macrophages and antibody ⁇ mediated cellular phagocytosis in vitro.
  • Tumors from humanized xenograft mice treated with NOA2 undergo growth arrest and contain intratumoral infiltrates of human macrophages.
  • A673, SKNEP ⁇ 1, and Kelly cells were cultured in DMEM (Gibco) and TC32, TTC466, RDES, TC71, and CADO ⁇ ES1 were cultured in RPMI (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin ⁇ streptomycin (Cellgro). Cells were incubated at 37oC with 5% CO2.293F cells were purchased from Thermo Fisher and cultured in Freestyle medium (Gibco). Cells were passaged a maximum of 8 ⁇ 10 times over 4 ⁇ 8 weeks.
  • Ewing sarcoma cells of various lines were incubated in a florescence ⁇ activated cell sorting (FACS) tube with 2 ⁇ g/mL of each anti ⁇ CD99 monoclonal antibody for one hour at 4oC followed by staining with a FITC ⁇ conjugated mouse anti ⁇ human IgG. Titration analyses were subsequently performed with NOA2 given that this candidate antibody demonstrated the strongest binding across Ewing lines.
  • FACS florescence ⁇ activated cell sorting
  • NOA2 and NOA3 were digested to form F(ab’)2 fragments such that the soluble CD99 ⁇ Fc can be immobilized on human Fc probes as the ligand while the F(ab’)2 fragments, at concentrates ranging from 0nM to 100100nM, can serve as the analyte.
  • ForteBio Octet software was utilized to analyze the data. Once binding curves were established, the experiment was repeated using analyte concentrations in a narrow range (10 ⁇ 15 nm for NOA2 and 10 ⁇ 25 nm for NOA3) so as to more accurately calculate each antibody KD.
  • ADCC Antibody ⁇ dependent cell ⁇ mediated cytotoxicity
  • ADCP Antibody ⁇ dependent cellular phagocytosis
  • PBMCs were plated in MDM medium (DMEM with 10% heat ⁇ inactivated human serum, 1% penicillin/streptomycin, 1% L ⁇ glutamine, and 50ng/mL GM ⁇ CSF) in a Petri dish for 3 days at 37oC with 5% CO2. At the end of three days, the supernatant was removed and MDM media replenished to further culture the adherent macrophage precursors. On day 7, macrophages were incubated with 0.5mM EDTA in PBS, lifted, and added at a ratio of 1:4 (target: effector cell) to Ewing sarcoma cells stained with PKH26 fluorescent membrane dye (Sigma Aldrich).
  • MDM medium DMEM with 10% heat ⁇ inactivated human serum, 1% penicillin/streptomycin, 1% L ⁇ glutamine, and 50ng/mL GM ⁇ CSF
  • Isotype IgG or NOA2 was added to the wells and incubated at 37oC for 4 hours. After co ⁇ culture, cells were imaged using a Celigo imaging cytometer (Nexcelom Biosciences) and then lifted and transferred to a 96 ⁇ well V ⁇ bottom plate for FACS. Cells were then counterstained with FITC ⁇ anti ⁇ CD14 for 30 minutes on ice. Engulfed target cells were defined as PKH26+/CD14+. ADCP was performed in duplicate for each cell line. Statistics were calculated using a two ⁇ tailed students t ⁇ test with a p value ⁇ 0.05 denoting significance.
  • ROIs regions of interest
  • mice were euthanized after 4 treatments, prior to the development of graft versus host disease, and tumor tissue harvested for hematoxylin eosin staining and immunohistochemistry.
  • Immunohistochemistry slides were imaged on a confocal microscope using a Nikon camera or the Keyence BZ ⁇ X800 system (Keyence Co. Itasca, IL).
  • Cells of various immune subtypes were counted from three 10x high ⁇ powered fields in representative tumors from mice treated with IgG or NOA2 and a DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 student’s two ⁇ tailed t ⁇ test (with significance denoted by a p ⁇ value ⁇ 0.05) run to compare counts.
  • monocytes were diluted to a concentration of 3 x10 6 cells/ml in complete RPMI ⁇ 1640 (Thermo Fisher), supplemented with 10% FBS, 100U/ml Penicillin/Streptomycin, 4.5g/L D ⁇ glucose, 2.383g/L HEPES, L ⁇ Glutamine, 15g/L sodium bicarbonate, and 110mg/L sodium pyruvate.3 x 10 4 cells were inserted in the upper chamber of a 96 ⁇ well TC ⁇ treated HTS Transwell Plate (Corning, CLS3388) with a 5.0 ⁇ m pore polycarbonate membrane.
  • a chemotactic gradient was established using different concentrations of MCP ⁇ 1 (monocyte chemoattractant protein ⁇ 1) or IGF ⁇ 1 (0.5 to 100ng/mL) in media supplemented with 8 ⁇ M Hoechst dye (Thermo Fisher). After 4 hours, the inserts were removed. Cells were spun down and the total fluorescent count from each well, as a reflection of cell migration, was recorded using a Celigo Imaging DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 Cytometer (Nexcelom Bioscience, LLC). These experiments were performed in triplicate and statistical significance calculated using a paired two ⁇ tailed student’s t ⁇ test with significance denoted by a p ⁇ value ⁇ 0.05.
  • PILR ⁇ synthesis and analysis of binding to human CD99 [00368] Human His ⁇ tagged PILR ⁇ was synthesized by cloning the extracellular domain into a pcDNA3.4 vector and transfecting Expi293F cells (Life Technologies). Protein was isolated from the supernatant and purified utilizing a HisPur Ni ⁇ NTA column (Qiagen). An SDS page gel demonstrated a band at 55kDa.
  • Protein synthesis was subsequently confirmed by ELISA, coating a plate with 2 ⁇ g/mL of our synthesized PILR ⁇ protein and incubating with a commercially available anti ⁇ human PILR ⁇ antibody (Sigma Aldrich) at a concentration of 2 ⁇ g/mL to assure binding. After verification, binding of human PILR ⁇ to human CD99 was assessed by ELISA. An ELISA plate was coated with 2 ⁇ g/mL of human CD99 and incubated with PILR ⁇ at concentrations ranging from 0.25 to 5 ⁇ g/mL and an anti ⁇ PILR ⁇ antibody with HRP ⁇ bound secondary.
  • Macrophage function in the context of PILR ⁇ and CD99 blockade Macrophage function in the context of PILR ⁇ and CD99 blockade
  • Monocyte ⁇ derived macrophages were isolated from PBMCs and identified as the cells left adherent following 5 ⁇ 7 days of PBMC culture. They were primed with 50ng/mL IFN ⁇ (Novus Biologicals) and triggered for M1 polarization with 10ng/mL lipopolysaccharide (LPS, Sigma Aldrich). 24 Twenty ⁇ four hours after LPS treatment, control group macrophages were incubated with one of three antibody conditions: anti ⁇ CD99 alone, anti ⁇ PILR ⁇ alone, or the combination of both antibodies.
  • the experimental group macrophages were co ⁇ incubated with CD99+ TC32 Ewing cells at a ratio of 2:1 (macrophage:Ewing cell) or Kelly CD99 ⁇ neuroblastoma cells and subsequently treated with each of the three antibody conditions.
  • This experiment was performed with Ewing cells on two separate occasions using different macrophage human donors and in duplicate, using a third donor, with Kelly neuroblastoma cells. Twelve hours later the supernatant was collected.
  • ELISA was performed on the supernatant to assess changes in the concentration of TNF ⁇ as a marker of macrophage activation (Abcam).
  • Phagemid panning for identification of anti-CD99 scFv’s Phagemid panning, utilizing a 27 billion member human single-chain variable fragment (scFv)-phage library, was performed for three rounds against immunotube-bound human CD99-Fc (G&P Biosciences). NUNC immunotubes were coated with soluble CD99 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 (10 ⁇ g/mL) and incubated at 4°C overnight. They were subsequently washed with phosphate- buffered saline (PBS) and blocked with 4% milk/PBS at 37°C for 1-2 hours.
  • PBS phosphate- buffered saline
  • TG1 bacteria were grown in 2xYT media in a baked flask at 37°C, shaking at 250 rpm until growth was recorded at an OD600 of 0.5-0.7.
  • the phagemid library was prepared for panning by diluting 5 x 1012 particles/mL in 2% milk/PBST (PBS-Tween), supplementing with isotype IgG to mitigate against binding to the Ig portion of CD99 Fc.
  • tubes were washed with PBS, loaded with 1 mL of the phage sample, and incubated for 90 minutes on a rotating platform and then stationary, for 30 minutes, at room temperature. Immunotubes were washed and bound-phage eluted using triethylamine.
  • Eluted phage were neutralized with 1M Tris and used to infect TG1 cells. Serial dilutions of infected bacterial cultures were plated and grown overnight at 37°C, while the remainder of the culture was plated on a 15 cm bacterial dish and grown overnight at 30°C. The next morning, bacteria were harvested from the 15 cm dish and grown until an OD of 0.5-0.7 was achieved. A volume of 10 mL of this culture was infected with VCS-M13 helper phage at a multiplicity of infection (MOI) of 20. Bacterial cells were resuspended in 2xYT containing ampicillin and kanamycin, grown overnight, and centrifuged the following morning.
  • MOI multiplicity of infection
  • Each unique clone was assessed for binding to membrane-associated CD99 on patient-derived Ewing sarcoma cell lines by fluorescence- activated cell sorting (FACs).
  • FACs fluorescence- activated cell sorting
  • the resultant cell-binding scFv’s were screened by ELISA against serial dilutions of soluble CD99 to identify the top three clones (NOA1, 2, and 3) with reproducible differential binding at concentrations of 0.5 ⁇ g/mL or less.
  • clone NOA1 forward 5’ – ATC CCA AGC TTA AGC CAG TCT GTG CTG AC T-3’
  • clone NOA2 forward 5’ – ATC CCA AGC TTA AGC CTG CCT GTG CTG ACT-3’
  • clone NOA3 forward 5’ – ATC CCA AGC TTA AGC TCC TAT GAG CTG ACT-3’.
  • the reverse primers used for light chain cloning were as follows: clone NOA1 reverse: 5’- GCT GAC CTA GGA GGA CGG TGA CCT T-3’ and clones NOA2 and NOA3 reverse: 5’- GCT GAC CTA GGA GGA CGG TCA GCT T- 3’.
  • Flow cytometric analysis of CD47 expression [00381] CD47 Ewing cell surface expression was analyzed by FACs for each Ewing sarcoma cell line at baseline and following treatment with NOA2 antibody.
  • Ewing sarcoma cells of various lines were incubated in a FACs tube alone or with varying concentrations of NOA2 antibody (range: 0.125 to 2.0 ⁇ g/mL) for one hour and secondarily stained with 1.0 ⁇ g/mL of anti-CD47 FITC bound antibody (ThermoFisher Scientific). Percentage of CD47-expressing cells, as a function of pre-incubation NOA2 concentration, was tabulated.
  • NOA2 and NOA3 were digested to form F(ab’)2 fragments such that the soluble CD99-Fc can be immobilized on human Fc probes as the ligand, while the F(ab’)2 fragments, at concentrates ranging from 0nM to 100nM, can serve as the analyte.
  • NOA1 was omitted from this assay due to low-affinity binding across Ewing lines by FACs.
  • ForteBio Octet software was utilized to analyze the data. Once binding curves were established, the experiment was repeated using analyte concentrations in a narrow range (10-15 nm for NOA2 and 10-25 nm for NOA3) so as to more accurately calculate each antibody KD.
  • NOA1 blue
  • NOA2 red
  • 3 green
  • NOA2 was carried forth for further studies given superior binding across Ewing cell lines.
  • the relative affinity of NOA2 was determined by flow cytometric saturation binding studies against five independent CD99 ⁇ positive Ewing sarcoma cell lines (FIG.24B). NOA2 binding was specific for CD99 ⁇ positive Ewing cells and demonstrated circa 3 ⁇ fold range in binding affinity across five Ewing lines.
  • FIG.24C demonstrates the heavy and light chain sequences and comparative human germlines for the NOA2 scFv.
  • ADCP was most prominent visually following incubation with 2 ⁇ g/mL indicating that lower concentrations of NOA2 can be more effective for this immune effector function.
  • NOA2 kills Ewing cells by both ADCC and ADCP, with differing efficiency across cell lines, implicating immune cells in antibody ⁇ mediated cytotoxicity.
  • FIG.26A demonstrates tumor growth arrest at 14 days post-treatment, as extrapolated by mean luminescence, in mice treated with NOA2 as compared with an isotype control.
  • NOA2’s mode of action mice were euthanized following therapy and livers dissected, embedded in paraffin, and sectioned (FIG.26B).
  • the representative images demonstrate a more prominent human CD45, mouse CD14, and human CD33 and CD16 infiltrate.
  • Anti ⁇ CD99 treated tumors also demonstrated pockets of necrosis indicative of apoptosis by TUNEL staining (FIG.26B).
  • FIG.26B The observation that treatment with NOA2 led to an accumulation of hCD45+ and hCD33+ immune cells in Ewing tumors in the PBL ⁇ NSG mouse study described herein led us to perform a second treatment study in CD34+ HSC reconstituted NSG ⁇ SGM3 mice which show enhanced engraftment of human myeloid cells.
  • 25,26 Mice were treated twice weekly with IgG or NOA2 and subcutaneous tumors harvested from mice at baseline and from each cohort following treatments 2, 4 and 5.
  • Human CD45+ cells were isolated from the harvested subcutaneous tumors and analyzed by FACS.
  • FIG.26D Tumors from the isotype or NOA2 treatment cohorts were grouped to allow for statistical analyses. As shown in FIG.26E there is a robust human CD33+ myeloid cell infiltrate in NOA2 ⁇ treated mice with ⁇ 55% of hCD45+ cells being derived from the myeloid lineage (p ⁇ 0.01). In summary, these collective results demonstrate that NOA2 treatment of humanized mice bearing Ewing tumors leads to the recruitment of CD33+ monocytes/myeloid cells.
  • Monocyte trafficking was analyzed in a transwell assay DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 utilizing a chemotactic gradient of MCP ⁇ 1 (positive control, FIG.27B left) or IGF ⁇ 1 (FIG. 27B right).
  • PILR ⁇ is a CD99 ligand, and inhibitory receptor, that is preferentially expressed on human myeloid cells but also on natural killer cells, dendritic cells, and other granulocytes. PILR ⁇ has been implicated in and can be restrictive of immune cell recruitment and activation. 27 ⁇ 30 Mouse PILR ⁇ has been shown to bind mouse CD99 but this protein interaction has not been well defined in humans. 31 As shown in FIG.28A we confirmed dose ⁇ dependent binding of human PILR ⁇ to soluble human CD99 by ELISA.
  • ELISA was first performed against an isotype-matched human DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 monoclonal IgG1 to subtract out Fc-region binders, and subsequently against soluble CD99- Fc.
  • DNA sequence analysis revealed that 10 were unique.
  • Six of these phagemid scFvs bound to A673 Ewing sarcoma cells by FACs. Binding affinity for these six scFv’s was assessed by ELISA with titration of soluble CD99-Fc concentrations.
  • the top three clones, NOA1, 2, and 3 demonstrated saturable binding at concentrations of 0.125 ⁇ g/mL or less.
  • NOA2 can trigger antibody ⁇ mediated tumor death through ADCC (Fc ⁇ R ⁇ mediated killing) and ADCP (phagocytosis).
  • ADCC Fe ⁇ R ⁇ mediated killing
  • ADCP phagocytosis
  • NOA2 is also associated with upregulation of IGF ⁇ 1, potentially contributing to monocyte/macrophage chemotaxis and recruitment.
  • A673 cells harbor a BRAFV600E mutation which is unusual in Ewing sarcoma and can have an atypical driver role besides the EWS fusion gene.
  • 32 We attempted to reconcile the differences in ADCP killing by examining the potential overexpression of CD47, recognizing that the CD47/SIRP ⁇ axis has been implicated in the control of myeloid cell activation.
  • 33 At baseline, human Ewing ⁇ derived cell lines express CD47, however, we could not demonstrate a clear upregulation of CD47 on A673 cells following treatment with NOA2 at concentrations of 1 ⁇ g/mL or higher (FIG.29).
  • CD99 antibody blockade has been shown to trigger Ewing cell death through upregulation of the IGF ⁇ 1R/Ras/Rac1 signaling pathway and a process termed methosis. 22 While our data corroborates upregulation of this pathway following binding of NOA2 therapy, NOA2 does not trigger Ewing cell death in the absence of immune cell engagement. This prompted us to evaluate whether the IGF ⁇ 1R/Ras/Rac1 signaling pathway serves a different purpose following NOA2 binding. The EWS ⁇ FLI1 fusion protein has been shown to activate the IGF1 promotor and induce IGF ⁇ 1 expression.
  • NOA2 can serve as an agonist on Ewing cells, upregulating EWS ⁇ FLI1 leading to enhanced IGF ⁇ 1 transcription (FIG.30).
  • IGF ⁇ 1 is upregulated in the tumors of NOA2 ⁇ treated mice and that this protein can serve as a monocyte chemoattractant (FIG.27) explaining the more prominent myeloid infiltrate visualized in NOA2 ⁇ vs, IgG ⁇ treated tumors from our in vivo studies.
  • Mouse CD14+ cells from our initial in vivo experiment appeared morphologically activated, similar to those seen in our ADCP DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 studies.
  • Menck K, Behme D, Pantke M, et al Isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon ⁇ coated cell culture bags. J Vis Exp:e51554, 2014 [00439] 24.

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Abstract

The treatment for pediatric solid tumor patients traditionally consists of a combination of chemotherapy, surgery, and radiation therapy. Targeted therapies, designed specifically for individual tumors, allow a more focused approach to treatment aimed at improving overall survival and diminishing side effects. This invention is directed to antibodies against CD99 and methods of use thereof.

Description

DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 ANTIBODIES AGAINST CD99 AND METHODS OF USE THEREOF [0001] This application claims priority to U.S. Provisional Application No.63/551,938 filed on February 09, 2024, the entire contents of each of which are incorporated herein by reference. [0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. [0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights. FIELD OF THE INVENTION [0004] This invention is directed to antibodies against CD99 and methods of use thereof. BACKGROUND OF THE INVENTION [0005] The treatment for pediatric solid tumor patients traditionally consists of a combination of chemotherapy, surgery, and radiation therapy. While these modalities are successful in curing patients with localized disease they are associated with substantial acute and long-term toxicities. Patients with widely metastatic or recurrent disease are rarely cured. Targeted therapies, designed specifically for individual tumors, allow a more focused approach to treatment aimed at improving overall survival and diminishing side effects. SUMMARY OF THE INVENTION [0006] Aspects of the invention are directed anti-CD99 antibody compositions. In one embodiment, the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, light chain, or a combination thereof. In some embodiments, the heavy chain comprises CDR1 comprising GYSFTNYW (SEQ ID NO: 19), GYSF-(X1X2X3)-W (SEQ ID NO: 13), GYTFTSYY (SEQ ID NO: 47), DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 GFSFSNYG (SEQ ID NO: 51), or GFTF-(X9X10X11)-G (SEQ ID NO: 54); CDR2 comprising IYPGDSDT (SEQ ID NO: 14), INPSGGST (SEQ ID NO: 48), I-( X5)-YDG-(X6X7X8) (SEQ ID NO: 52), or VSHDDINK (SEQ ID NO: 55); and/or CDR3 comprising ARRANPGAFDI (SEQ ID NO: 16), ARPCSAGSCYSTDAFDI (SEQ ID NO: 15), , ARHLRNFGDAFDI (SEQ ID NO: 17), ARDLSSNGDY (SEQ ID NO: 49), ARHLRHFGDAFDV (SEQ ID NO: 50), AR-(X12X13X14)-MDV (SEQ ID NO: 53), or AKGYSGTYGIYFDY (SEQ ID NO: 56). In other embodiments, the light chain comprises CDR1 comprising SDINVGSYN (SEQ ID NO: 23), KLGNTY (SEQ ID NO: 20), ALPKQY (SEQ ID NO: 26), SSDVGGYNY (SEQ ID NO: 55), SGSIATN-(X15) (SEQ ID NO: 58), SLRSYY (SEQ ID NO: 63), or SSNIGSNY (SEQ ID NO: 66); CDR2 comprising YYSDSNK (SEQ ID NO: 24), QD(X4) (SEQ ID NO: 28), DVS (SEQ ID NO: 56), KDS (SEQ ID NO: 98), EDN (SEQ ID NO: 59), KDD (SEQ ID NO: 61), GKN (SEQ ID NO: 64), RNN (SEQ ID NO: 67); and/or CDR3 comprising MIWHSSAWV (SEQ ID NO: 25), QAWDSSTAV (SEQ ID NO: 22), FSYAGDNRGV (SEQ ID NO: 57), QSYDRTNSVVL (SEQ ID NO: 60), QSYDDGSHYV (SEQ ID NO:62), SSRDSSGNHPYV (SEQ ID NO: 65), or AAWDGSLSGYV (SEQ ID NO: 68). In embodiments, X1, X2, X3, or X4 is a hydrophilic polar amino acid residue. In embodiments, the hydrophilic polar amino acid residue is Threonine (T), Asparagine (N), Lysine (K), Histidine (H), or Serine (S). In embodiments, X1 is a neutral non-polar amino acid residue, for example the amino acid residue is Proline (P) or Tyrosine (Y). In embodiments, X1X2X3 is STH, PSH, TNY, or SKY. In embodiments, X4 is S. In embodiments, X4 is N. In embodiments, X9 or X10 is a hydrophilic polar amino acid residue, for example the amino acid residue is Asparagine (N), Serine (S), Threonine (T), Arginine (R). In embodiments, X11 is a neutral non-polar amino acid residue, for example the amino acid residue is Phenylalanine (F) or Tyrosine (Y). In embodiments, X9X10X11 is RNY, NTF, or SSY. In embodiments, X5, X6, X7, or X8 is a hydrophilic polar amino acid residue, for example the amino acid residue is Asparagine (N), Serine (S), Lysine (K), Histidine (H), Arginine (R), or Glutamate (E). In embodiments, X5 or X8 is a non-polar amino acid residue, for example the amino acid residue is Phenylalanine (F) or Leucine (L). In embodiments, X5 is Serine (S) or Phenylalanine (F). In embodiments, X6X7X8 is HKE, SNK, or SRL. In embodiments, X12 or X13 is a hydrophilic polar amino acid residue, for example the amino acid residue is Serine (S) or Arginine (R). In embodiments, X13 or X14 is a hydrophobic, non-polar amino acid residue, for example the amino acid residue is phenylalanine (F) or glycine (G). In embodiments, X12X13X14 is RGF or SRG. In embodiments, X15 is a non-polar amino acid residue, for example X15 phenylalanine (F), or tyrosine (Y). In some embodiments, the antibody is fully human. In DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 embodiments, the antibody is monospecific or bispecific. In embodiments, the bispecific target is LINGO1 or IGF1R. In embodiments, the antibody is a single chain antibody. In embodiments, the antibody is an IgG. In embodiments, the antibody has a binding affinity of at least 1.0 x10-9 M. In embodiments, the antibody is linked to a therapeutic agent. In embodiments, the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine. [0007] An aspect of the invention is directed to an antibody composition comprising at least one antibody, wherein the at least one antibody comprises two heavy chains and two light chains. In some embodiments, the heavy chain CDRs are identical to reference germline CDRs found between residues 26 and 33, residues 51 and 58, and residues 97 and 113 according to IMGT numbering of SEQ ID NO: 29, or between residues 97 and 108 according to IMGT numbering of SEQ ID NO: 33, or between residues 97 and 110 according to IMGT numbering of SEQ ID NO: 34, except that at least one of the heavy chain CDRs differs by a single amino acid substitution relative to its reference CDR. In other embodiments, the light chain CDRs are identical to reference germline CDRs found between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:30, or between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:110 or between residues 26 and 34, residues 52 and 58, and residues 97 and 104 according to IMGT numbering of SEQ ID NO: 31, except that at least one of the light chain CDRs differs by a single amino acid substitution relative to its reference CDR. In further embodiments, the antibody composition binds to an epitope that comprises amino residues at positions 23 to 123 of the extracellular domain of CD99 comprising SEQ ID NO: 32. In embodiments, the antibody is fully human. In embodiments, the antibody is a monoclonal antibody. In embodiments, the antibody is an IgG. In embodiments, the antibody is monospecific or bispecific. In embodiments, the antibody has a binding affinity of at least 1.0 x10-9 M. In embodiments, the heavy chain CDRs include one or more of the following residue substitutions: 30Pro or 30 or 31Ser; 31Asn or 31or 32Lys; or 32His relative to SEQ ID NO: 29 or SEQ ID NO: 34; or 103Gly relative to SEQ ID NO: 33. In embodiments, the light chain CDRs include one or more of the following residue substitutions: 26Ala; 28Pro; 29Asn or 29Lys; 30Thr or 30Gln; 51Asn relative to SEQ ID NO: 30; or 57Asn; 100His; 102Ser; or 104Trp relative to SEQ ID NO: 31. [0008] An aspect of the invention is directed to a pharmaceutical composition comprising: one or more antibody compositions comprising at least one antibody, wherein the at least one antibody comprises two heavy chains and two light chains. In some embodiments, the heavy DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 chain CDRs are identical to reference germline CDRs found between residues 26 and 33, residues 51 and 58, and residues 97 and 113 according to IMGT numbering of SEQ ID NO: 29, or between residues 97 and 108 according to IMGT numbering of SEQ ID NO: 33, or between residues 97 and 110 according to IMGT numbering of SEQ ID NO: 34, except that at least one of the heavy chain CDRs differs by a single amino acid substitution relative to its reference CDR. In other embodiments, the light chain CDRs are identical to reference germline CDRs found between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:30, or between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:110 or between residues 26 and 34, residues 52 and 58, and residues 97 and 104 according to IMGT numbering of SEQ ID NO: 31, except that at least one of the light chain CDRs differs by a single amino acid substitution relative to its reference CDR. In further embodiments, the antibody composition binds to an epitope that comprises amino residues at positions 23 to 123 of the extracellular domain of CD99 comprising SEQ ID NO: 32. In embodiments, the heavy chain CDRs include one or more of the following residue substitutions: 30Pro, 32His of SEQ ID NO: 29 relative to SEQ ID NO: 40. In embodiments, the heavy chain CDRs include one or more of the following residue substitutions: 31Asn, 103Gly of SEQ ID NO: 33 relative to SEQ ID NO: 40. In embodiments, the heavy chain CDRs include one or more of the following residue substitutions: 30 or 31Ser; 31or 32Lys of SEQ ID NO: 34 relative to SEQ ID NO: 40. In embodiments, the light chain CDRs include one or more of the following residue substitutions: 29Asn; 30Thr; or 51Asn of SEQ ID NO: 30 relative to SEQ ID NO: 41. In embodiments, the light chain CDRs include one or more of the following residue substitutions: 26Ala; 28Pro; 29Lys; or 30Gln of SEQ ID NO: 110 relative to SEQ ID NO: 41. In embodiments, the light chain CDRs include one or more of the following residue substitutions: 57Asn; 100His; 102Ser; or 104Trp of SEQ ID NO: 31 relative to SEQ ID NO: 42. In embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient. In embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent. In embodiments, the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine. [0009] An aspect of the invention is directed to a cell producing an antibody described herein. [0010] An aspect of the invention is directed to a genetically engineered cell, wherein the genetically engineered cell expresses and/or secretes an antibody described herein. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [0011] An aspect of the invention is directed to methods of treating cancer. In some embodiments, the method comprises administering to a subject in need thereof a composition comprising an antibody described herein or pharmaceutical composition described herein. In some embodiments, the cancer expresses CD99. In embodiments, the cancer comprises Ewing Sarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, astrocytoma, glioblastoma, pancreatic endocrine tumor, GI/pulmonary neuroendocrine tumor, prostate cancer, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, lymphoma, or a combination of the CD-99 expressing cancers described herein. [0012] An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:3, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:4. [0013] An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:7, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:8. [0014] An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:11, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:12. [0015] An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:71, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:72. [0016] An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:75, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:76. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [0017] An aspect of the invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:79, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:80. [0018] An aspect of the invention is directed to a nucleic acid encoding an antibody described herein. In embodiments, the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 5 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 6; the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 1 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 2; or the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 9 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 10. In embodiments, the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 69 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 70; the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 73 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 74; or the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 77 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 78. [0019] An aspect of the invention is directed to a vector comprising the nucleic acid encoding an antibody described herein. [0020] An aspect of the invention is directed to a cell comprising the vector comprising the nucleic acid encoding an antibody described herein. [0021] An aspect of the invention is directed to a kit comprising at least one antibody composition described herein; a syringe, needle, or applicator for administration of the at least one antibody or fragment thereof to a subject; and instructions for use. [0022] An aspect of the invention is directed to a probe comprising an antibody described herein, and wherein the antibody is further coupled to an imaging agent. [0023] An aspect of the invention is directed to an isolated anti-CD99 antibody comprising an antibody described herein, wherein the antibody competes with binding to CD99 or binds the same epitope as PILR^ or an anti-PILRα antibody. [0024] An aspect of the invention is directed to a chimeric antigen receptor (CAR) cell that expresses and secretes an antibody described herein. In embodiments, the antibody comprises an anti-CD99 antibody. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [0025] Other objects and advantages of this invention will become readily apparent from the ensuing description. BRIEF DESCRIPTION OF THE FIGURES [0026] FIG.1 shows the scFv binding affinity by ELISA as a function of soluble CD99 concentration (range: 0.3125μg/mL to 2μg/mL). [0027] FIG.2 shows FACs to assess anti-CD99 mAb binding of clone 2, at varying concentrations (range: 0.008μg/mL to 2μg/mL), to A673 Ewing sarcoma cells. The red histogram represents cells alone. [0028] FIG.3, panels A-C, demonstrate cell death of three human-derived Ewing sarcoma cell lines (CADO-ES1, TC32, A673) following incubation with anti-CD99 mAb clones and rabbit serum complement. Panels D-F demonstrate Ewing cell death following incubation with anti-CD99 mAb and Jurkat effector cells. [0029] FIG.4 shows representative markers, corresponding to tumor infiltrating immune cells, to be assessed by IHC and flow cytometry. [0030] FIG.5 shows a timeline of events leading to the creation of a humanized Ewing xenograft model. [0031] FIG.6 shows A) FACs demonstrating A673 Ewing sarcoma cells alone (red), A673 cells incubated with a non-specific FITC-bound isotype control (blue), A673 cells incubated with 10 µg/mL of NOA1 (orange), 2 µg/mL of NOA2 (light green), and 2 µg/mL of NOA3 (dark green). Three samples were incubated with a mouse anti-human FITC-conjugated secondary. Panel B shows FACs demonstrating Kelly neuroblastoma alone and in each of the conditions described herein. Panel C shows FACs demonstrating diminishing fluorescence with decreasing concentrations of NOA2 or 3 bound to A673 Ewing sarcoma cells. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [0032] FIG.7 shows OctetRED analysis for NOA2 (Panel A, NOA2 F(ab’)2 concentrations of 15nM (Sensor A), 13.5nM (Sensor B), 12.5nM (Sensor C), 11.4nM (Sensor D), 10nM (Sensor E), and 0 nM (Sensor F)) and for NOA3 (Panel B, NOA3 F(ab’)2 concentrations of 25nM (Sensor A), 22.5nM (Sensor B), 20nM (Sensor C), 17.5nM (Sensor D), 15nM (Sensor E), 12.5nM (Sensor F), 10nM (Sensor G), and 0nM (Sensor H). [0033] FIG.8 shows panels A-C, which demonstrate cell death of three human-derived Ewing sarcoma cell lines (CADO-ES1, TC32, A673) by ADCC following incubation with anti-CD99 mAb and Jurkat effector cells. Panels D-F demonstrate Ewing cell death by CDC following incubation with anti-CD99 mAb clones and rabbit serum complement. [0034] FIG.9 provides confocal microscopy images demonstrating NOA2 internalization. Panels A and F) demonstrate light-field images of a singular TC32 cell, Panels B and G) Hoescht nuclear staining, Panels C and H) DiD dye in a membranous distribution with pockets of dye in the cytoplasm, Panels D and I) FITC-bound NOA2 in a membranous distribution also with pockets of dye in the cytoplasm, and Panels E and J) co-localization of DiD and FITC-bound NOA2 throughout the cytoplasm indicating endocytosis-driven NOA2- propagated CD99 internalization. [0035] FIG.10 shows mean fluorescence measured by BLI for mice treated with an isotype control versus NOA2. Panel A demonstrates tumor growth arrest in mice treated with NOA2 antibody 24 hours following PBMC infusion. Panel B displays tumors from mice treated with IgG or NOA2 embedded in paraffin and stained with hematoxylin eosin, anti-CD99, and human anti-CD45, mouse anti-CD14, anti-IGF1R and Ras, and TUNEL to assess for apoptosis. [0036] FIG.11 shows panel A, which provides light microscopy images of macrophages actively engulfing Ewing sarcoma cells following incubation with NOA2 (labeled red with DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 PKH26). Panel B demonstrates a graphical representation of PKH26+/CD14+ cell count (i.e. Ewing cells engulfed by macrophages) following treatment with NOA2. [0037] FIG.12 shows panel A, which demonstrates ELISA results of PILR^:CD99 binding in the presence of varying concentrations of PILR^. Panel B shows PILR^ binding to A673 Ewing cells in the presence of different concentrations of anti-CD99 antibody. Panel C illustrates an interaction between macrophage PILR^ and Ewing cell CD99 and the role of anti-PILR^ or anti-CD99 antibodies in the disruption of the binding. Panel D demonstrates macrophage TNF-^ secretion following the dual incubation of macrophages and Ewing cells with anti-PILR^ and anti-CD99 antibodies. [0038] FIG.13 demonstrates the experimental design of cellular migration studies and associated data under one embodiment. [0039] FIG.14 shows heavy and light chain sequences and comparative human germlines for three binding anti-CD99 scFv’s (NOA1, 2, and 3). Heavy chain germline amino acid sequence is SEQ ID NO: 40. NOA1 heavy chain amino acid sequence that follows the germline sequence in the alignment is SEQ ID NO: 29. NOA2 heavy chain amino acid sequence that follows the NOA1 sequence in the alignment is SEQ ID NO: 33. NOA3 heavy chain amino acid sequence that follows the NOA2 sequence in the alignment is SEQ ID NO: 34. Light chain germline IGLV3-1*01 amino acid sequence is SEQ ID NO: 41. NOA1 light chain amino acid sequence that follows the germline sequence in the alignment is SEQ ID NO: 30. NOA3 light chain amino acid sequence that follows the NOA1 sequence in the alignment is SEQ ID NO: 110. Light chain germline IGLV5-37*01 amino acid sequence is SEQ ID NO: 42. NOA2 light chain amino acid sequence that follows the germline sequence in the alignment is SEQ ID NO: 31. [0040] FIG.15 depicts FACS graphs as well as EC50 graphs. Panel A (upper) depicts FACs for A673 Ewing sarcoma cells alone (purple), A673 cells incubated with a non-specific DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 FITC-bound isotype control (orange), A673 cells incubated with 10 µg/mL of NOA1 (blue), 2 µg/mL of NOA2 (red), and 2 µg/mL of NOA3 (green). Three samples were incubated with a mouse anti-human FITC-conjugated secondary. The lower panel depicts FACs for Kelly neuroblastoma alone and in each of the conditions described herein. Panel B depicts FACs demonstrating diminishing fluorescence with decreasing concentrations of NOA2 bound to A673, TC32, CADO-ES1, TC71 and TTC466 Ewing sarcoma cells. Panel C displays the EC50 generated from the geometric mean fluorescence to account for relative CD99 expression per cell line. [0041] FIG.16 shows graphs and light microscopy micrographs. Panels A-C demonstrate cell death of three human-derived Ewing sarcoma cell lines (CADO-ES1, TC32, A673) by ADCC following incubation with anti-CD99 mAb and Jurkat effector cells. Panel D shows light images obtained utilizing Celigo demonstrating aggregated, plump macrophages actively engulfing Ewing sarcoma cells following incubation with NOA2 (labeled red with PKH26), i.e. ADCP. This finding was more prominent for the p53 wildtype cell lines CADO-ES1 and TC32 (Panel A, arrows). Panel E demonstrates a dose-independent increase in PKH26+/CD14+ cells (i.e. Ewing cells engulfed by macrophages) following treatment with NOA2 in CADO-ES1 and TC32. [0042] FIG.17 shows mean fluorescence graphs as well as light microscopy images. Panel A depicts mean fluorescence measured by BLI for mice treated with an isotype control (n=2) versus NOA2 (n=3). Arrows correspond with timed mouse treatments. Panel B displays tumors from mice treated with IgG or NOA2 embedded in paraffin and stained with hematoxylin eosin, anti-CD99, human anti-CD45/CD33/CD16, mouse anti-CD14, anti-IGF- 1R, IGF-1, Ras, and TUNEL to assess for apoptosis. While tumors stain positive for CD99, only tumors from mice treated with NOA2 demonstrated TUNEL positivity, an activated mouse anti-CD14+ and human CD16+/CD33+ myeloid infiltrate, upregulation of IGF-1R DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 and Ras, and an increase in IGF-1. Panel C depicts the numbers of each infiltrating immune cell type counted in three 10x high-powered fields. [0043] FIG.18 shows graphs of chemotaxis experiments. Panel A indicates chemotaxis of human monocytes in a dose-dependent fashion through a transwell membrane towards IGF-1 (with MCP-1 included as a positive control). [0044] FIG.19 shows a graph of flow cytometry of the immune cell compartment of subcutaneous Ewing tumors grown in humanized mice demonstrate a robust CD33+ infiltrate. Grouping tumors from more than one NOA2 treatment timepoint (Panel A), allows a calculation of statistical significance between groups. [0045] FIG.20 shows graphs of binding assays. Human PILR^ binds to both plate-bound and Ewing cell-surface human CD99. Panel A demonstrates ELISA results confirming PILR^-CD99 binding in a PILR^-dose dependent fashion. Panel B shows that with escalating concentrations of anti-CD99 antibody, there is diminished PILR^ binding to A673 Ewing cells. Panel C shows the rebound in macrophage TNF-^ secretion detected following dual incubation of macrophages and Ewing cells with both anti-PILR^ and anti-CD99 antibodies. Panel D outlines the interaction between macrophage PILR^ and Ewing cell CD99 as well as the interruption in this binding with anti-PILR^ or anti-CD99 antibodies. [0046] FIG.21 shows graphs of cell surface expression. Panel A demonstrates cell surface expression of CD47 in CADO-ES1, TC32, and A673 following pre-treatment with NOA2. While three cell lines demonstrate an increase in CD47 expression following pre-treatment with varying doses of NOA2, A673 demonstrates the largest increase in expression at the highest concentrations of NOA2. [0047] FIG.22 shows a schematic of the downstream effects of NOA2 binding to membranous CD99 on Ewing sarcoma cells resulting in increased IGF-1 transcription and DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 secretion. IGF-1 then acts to further upregulate the IGF-1/IGF-1R pathway through an autocrine loop. [0048] FIG.23 shows sensorgrams of antibody binding. OctetRED analysis for NOA2 and 3. Panel A: NOA2 F(ab’)2 concentrations utilized were as follows: 15nM (Sensor A), 13.5nM (Sensor B), 12.5nM (Sensor C), 11.4nM (Sensor D), 10nM (Sensor E), and 0 nM (Sensor F). Panel B: NOA3 F(ab’)2 concentrations utilized were as follows: 25nM (Sensor A), 22.5nM (Sensor B), 20nM (Sensor C), 17.5nM (Sensor D), 15nM (Sensor E), 12.5nM (Sensor F), 10nM (Sensor G), and 0nM (Sensor H). [0049] FIG.24 shows FACs binding by IgG1 isoforms to CD99-positive patient-derived Ewing sarcoma cell lines. Panel A (left) shows FACS for A673 Ewing sarcoma cells alone (purple), A673 cells incubated with a non‐specific FITC‐bound isotype control (orange), A673 cells incubated with 10 μg/mL of NOA1 (blue), 2 μg/mL of NOA2 (red), and 2 μg/mL of NOA3 (green). Three samples were incubated with a mouse anti‐human FITC‐conjugated secondary. Panel A (right) shows FACS for Kelly neuroblastoma alone and in each of the conditions described herein. Panel B shows FACS demonstrating diminishing fluorescence with decreasing concentrations of NOA2 bound to A673, TC32, CADO‐ES1, TC71 and TTC466 Ewing sarcoma cells. Panel C shows OctetRED analysis for NOA2. NOA2 F(ab’)2 concentrations utilized were as follows: 15nM (Sensor A), 13.5nM (Sensor B), 12.5nM (Sensor C), 11.4nM (Sensor D), 10nM (Sensor E), and 0 nM (Sensor F). Panel D provides the heavy and light chain sequences, comparative to germline, for NOA2. [0050] FIG.25 Panels A‐C show cell death of three human‐derived Ewing sarcoma cell lines (CADO‐ES1, TC32, A673) by ADCC following incubation with anti‐CD99 mAb and Jurkat effector cells. Kelly neuroblastoma cells were used as a negative control. Panel D shows light images obtained utilizing Celigo demonstrating aggregated, plump macrophages actively engulfing Ewing sarcoma cells following incubation with NOA2 (labeled red with PKH26), DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 i.e. ADCP. This finding was more prominent for the p53 wildtype cell lines CADO‐ES1 and TC32 (Panel A, Panel D arrows). Panel E indicates a potential dose‐independent increase in PKH26+/CD14+ cells (i.e. Ewing cells engulfed by macrophages) following treatment with NOA2 in CADO‐ES1 and TC32 but this is not bourne out in graphical representation of the data (Panel F). [0051] FIG.26 Panel A shows mean fluorescence measured by BLI for mice treated with an isotype control (n=2) versus NOA2 (n=3). Arrows correspond with timed mouse treatments. Panel B shows tumors from mice treated with IgG or NOA2 embedded in paraffin and stained by immunohistochemistry. Tumors from mice treated with NOA2 demonstrate a more prominent human CD45+ infiltrate, human CD16+/CD33+ staining myeloid cells, and plump, activated‐appearing mouse CD14+ cells compared with tumors from mice treated with IgG. NOA2‐treated tumors also stain for TUNEL indicating apoptosis. Panel C shows the numbers of each infiltrating immune cell type counted in three 10x high‐powered fields. Panel D shows flow cytometry results of the immune cell infiltrate isolated from subcutaneous tumors in a follow‐up humanized mice experiment. Tumors were isolated from one mouse in each treatment cohort at each of the timepoints indicated. Tumors were grouped to allow a calculation of statistical significance between cohorts (Panel E). [0052] FIG.27 Panel A shows immunohistochemical staining of tumors from the micro‐ metastatic mouse model depicted in Figure 3A‐C. Tumors from mice treated with NOA2, as compared with mice treated with IgG, show upregulation of IGF‐1, Ras, and IGF‐1 implicating the IGF‐1/Ras‐Rac‐1 pathway downstream of CD99 binding. Panel B shows results to a chemotaxis assay investigating whether IGF‐1 is responsible for monocyte migration. Human monocytes migrate through a transwell membrane towards concentrations of IGF‐1 greater than 10 ng/mL. The same assay was performed using MCP‐1 as a positive control (left). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [0053] FIG.28 Panel A shows ELISA results confirming human PILRα binding to human CD99 in a PILRα‐dose dependent fashion. Panel B shows the interaction between macrophage PILRα and Ewing CD99 (left) as well as the potential inhibitory pathway triggered by binding both macrophage CD99 and PILRα (right upper). Panel B (right lower) shows disruption of the CD99: PILRα macrophage:Ewing checkpoint pathway. Panel C shows the rebound in macrophage TNF‐α secretion detected following co‐incubation of macrophages with Ewing cells and both anti‐PILRα and anti‐CD99 antibodies. [0054] FIG.29 provides heavy and light chain sequences and comparative human germlines for the top three binding anti-CD99 scFv’s (NOA1, 2, and 3). Red letters indicate single nucelotide mutations (SNMs). [0055] FIG.30 shows repeat FACS analysis to depict the geometric mean fluorescence of A673, TC71 and TTC466. [0056] FIG.31 shows CD47 in CADO-ES1, TC32, and A673 following pre-treatment with NOA2. Three cell lines demonstrate a modest increase in CD47 expression following pre- treatment with varying doses of NOA2. [0057] FIG.32 shows the downstream effects of NOA2 binding to membranous CD99 on Ewing sarcoma cells resulting in increased IGF-1 transcription and secretion which then acts to further upregulate the IGF-1/IGF-1R pathway through an autocrine loop. [0058] FIG.33 shows cell surface expression of CD99, detected by NOA2 binding, on monocytes derived from human peripheral blood mononuclear cells. Panel A shows PBMC separated into lymphocyte and monocyte populations (depicted within the superimposed square) based on side and forward scatter. Monocytes were unstained (Panel B) and incubated with anti-human FITC-bound secondary (Panel C). Panel C shows non-specific binding of the anti-human FITC-bound secondary to monocytes, which is accounted for in DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 gating of cells in Panel D. The population of CD33+ cells that express CD99 (by NOA2 binding) are depicted in Panel D. DETAILED DESCRIPTION OF THE INVENTION [0059] Abbreviations and Definitions [0060] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner. [0061] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” [0062] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting. [0063] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited. [0064] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context. [0065] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower). [0066] Aspects of the invention provide isolated monoclonal antibodies specific against CD99. The term “isolated” as used herein with respect to cells, nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term “isolated” can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. For example, an “isolated nucleic acid” can include nucleic acid fragments which are not naturally occurring as fragments and cannot be found in the natural state. “Isolated” can also refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides can include both purified and recombinant polypeptides. The antibodies were identified through the use of a 27 billion human single- chain antibody (scFv) phage display library, by using soluble human CD99 as a library selection target. These antibodies represent a new class of monoclonal recombinant antibodies against CD99, for example, see Tables 1-11. “Recombinant” as it pertains to polypeptides (such as antibodies) or polynucleotides can refer to a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that cannot normally occur together. [0067] The CD99 antibodies described herein bind to CD99. In one embodiment, the CD99 antibodies have high affinity and high specificity for CD99. Some embodiments also feature antibodies that have a specified percentage identity or similarity to the amino acid or nucleotide sequences of the anti-CD99 antibodies described herein. For example, “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity when compared a specified region or the full length of any one of the anti-CD99 antibodies described herein. For example, the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 higher nucleic acid identity when compared to a specified region or the full length of any one of the nucleic acid sequences encoding anti-CD99 antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and/or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e. BLAST or BLAST 2.0), manual alignment or visual inspection can be utilized to determine percent sequence identity or similarity for the nucleic acids and proteins of the invention. [0068] “Polypeptide” as used herein can encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed 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 the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, can refer to “polypeptide” herein, and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. “Polypeptide” can also refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a nucleic acid sequence. It can be generated in any manner, including by chemical synthesis. As to amino acid sequences, one of skill in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence is collectively referred to herein as a "conservatively modified variant". In some embodiments the alteration results in the substitution of an amino acid with a chemically similar amino acid. [0069] As to amino acid sequences, one of skill in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence is collectively referred to herein as a "conservatively modified variant". In some embodiments the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 conservatively modified variants of the anti-CD99 antibodies disclosed herein can exhibit increased cross-reactivity to CD99 in comparison to an unmodified CD99 antibody. [0070] For example, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, 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), nonpolar 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 (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. [0071] Antibodies [0072] As used herein, an “antibody” or “antigen-binding polypeptide” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. For example, “antibody” can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Non-limiting examples a complementarity determining region (CDR) of a heavy or light chain 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 portion of a binding protein. As used herein, the term "antibody" can refer to an immunoglobulin molecule and immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen. By "specifically binds" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the antigen and does not react with other polypeptides. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, antigen-binding fragments (Fab), Fab' and F(ab')2 fragments, scFvs, and Fabexpression libraries. [0073] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab′)2, F(ab)2, Fab′, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 antibody. The term “antibody fragment” can include aptamers (such as spiegelmers), minibodies, and diabodies. The term “antibody fragment” can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanizedor chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, dAb (domain antibody), minibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. [0074] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. A single chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide- encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. A number of methods have been described to discern chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule, which will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Patent Nos.5,091,513; 5,892,019; 5,132,405; and 4,946,778, each of which are incorporated by reference in their entireties. [0075] Very large naive human scFv libraries have been and can be created to offer a large source of rearranged antibody genes against a plethora of target molecules. Smaller libraries can be constructed from individuals with infectious diseases in order to isolate disease- specific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al, Proc. Natl. Acad. Sci. USA 89:3175-79 (1992)). [0076] Antibody molecules obtained from humans fall into five classes of immunoglubulins: IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain in the molecule. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 among them (e.g., γ1-γ4). Certain classes have subclasses as well, such as IgG1, IgG2, IgG3 and IgG4 and others. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and are known to confer functional specialization. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region. Immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of an immunoglobulin molecule. [0077] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can be bound with a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated by hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. [0078] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. The variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. The term "antigen-binding site," or "binding portion" can refer to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus, the term "FR" can refer to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs." VH and VL regions, which contain the CDRs, as well as frameworks (FRs) of the CD99 antibodies (e.g., scFvs) are shown in Tables 1A-10B. [0079] The nucleic acid and amino acid sequence of the monoclonal human anti-CD99 antibodies are provided herein: Table 1A. Ab #NOA1 Variable Region nucleic acid sequences VH chain of Ab #AO1 (IGHV5-51*03, IGHD2-15*01, IGHJ3*02) g gt c tc ta ct c ab e . b O ar ab e eg on am no ac d sequences VH chain of Ab #AO1 (IGHV5-51*03, IGHD2-15*01, IGHJ3*02) Q D S Table 2A. Ab #NOA2 Variable Region nucleic acid sequences c gt c ta tc ct Table 2B. Ab #NOA2 Variable Region amino acid sequences DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 QVQLVQSGPEVKKPGESLKISCEGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQ GQVTISADKSISSAYLQWSSLQASDTGMYYCARRANPGAFDIWGQGTMVTVSS (SEQ ID NO: 7) S VH chain of Ab #AO3 (IGHV5-51*01, IGHD3-16*01, IGHJ3*02) ca a a t ct g . g q VH chain of Ab #AO3 (IGHV5-51*01, IGHD3-16*01, IGHJ3*02) Q ) S Table 4A. Ab NOA4 Variable Region nucleic acid sequences VH chain of Ab NOA4 (IGHV1-46*01/03 IGHD6-13*01 IGHJ4*02/03) ct g a gt a gc gc Table 4B. Ab NOA4 Variable Region amino acid sequences * * * F ) DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFS GSKSGNTASLTVSGLQAEDEADYYCFSYAGDNRGVFGAGTQVTVL (SEQ ID NO: 72) VH chain of Ab NOA5 (IGHV1-46*01/03, IGHD6-13*01, IGHJ4*02/03) ct g a gt tt c g 4) VH chain of Ab NOA5 (IGHV1-46*01/03, IGHD6-13*01, IGHJ4*02/03) F ) S VH chain of Ab NOA6 (IGHV5-51*01 IGHD3-16*01 IGHJ3*01) ct at c ca aa tc ta a e . ara e egon amno ac sequences V hi f A N A I HV 1*1 I HD 1*1 I H *1 V S Table 7A. Ab NOA7 Variable Region nucleic acid sequences DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 gaggtgcagctggtggagtctgggggaggcgtagtccagcctgggaggtccctgagacttacctgtgcagcctctggattctcctt cagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtgacaggcatatcatatgatggacataa t t t t tt tt tt tttt t g tc t tg g D VH chain of Ab NOA7 (IGHV3-30*03/18, IGHD3-10*01, IGHJ6*03) V ) SI VH chain of Ab NOA8 (IGHV3-30*03, IGHD1-26*01, IGHJ4*02) t ta tg ac a tt g . V chain of Ab NOA8 (IGHV3-30*03 IGHD1-26*01 IGHJ4*02) V S Table 9A. Ab NOA9 Variable Region nucleic acid sequences * * tt at cc DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 tgagagccgaggacacggctgtgtattactgtgcgagatcacgcggtatggacgtctggggccaagggaccacggtcaccgtctc ctca (SEQ ID NO: 89) * * tg g tg VH chain of Ab NOA9 (IGHV3-30*03, unknown, IGHJ6*02) S S . VH chain of Ab NOA10 (IGHV3-33*03, IGHD3-10*01, IGHJ6*03) tt a ct cc t a gg Table 10B. Ab NOA10 Variable Region amino acid sequences VH chain of Ab NOA10 (IGHV3-33*03 IGHD3-10*01 IGHJ6*03) V ) S [0080] Table 11. Amino Acid Sequences of Heavy and Light Chain CDRs. Antibody Variable i CDR1 CDR2 CDR3 DI DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 Antibody Variable region CDR1 CDR2 CDR3 [0081] The six CDRs in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, the FR regions, show less inter- DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. The framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for a heavy or light chain variable region by one of ordinary skill in the art, since they have been previously defined (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)). [0082] Where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol.196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table herein as a comparison. The exact residue numbers which encompass a CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a CDR given the variable region amino acid sequence of the antibody. CDR Kabat Numbering Chothia Numbering DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 VL CDR3 89-97 91-96 [ iable domain sequences that is applicable to any antibody. The skilled artisan can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). [0084] In addition to table herein, the Kabat number system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR- H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at 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 (i.e., following a cysteine residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid. [0085] In one embodiment, VH CDR1 can be GYSF -X1X2X3-W (SEQ ID NO: 13). In some embodiments, X1, X2, X3, or X4 can be a hydrophilic polar amino acid residue (e.g., Threonine (T), Asparagine (N), or Serine (S), Lysine (K), Histidine (H)). In some embodiments, X1 can be a neutral non-polar amino acid residue (e.g., proline (P) Tyrosine (Y)). [0086] In one embodiment, VL CDR2 can be QD-X4 (SEQ ID NO: 28). In some embodiments, X4 can be a hydrophilic polar amino acid residue (e.g., Asparagine (N), or Serine (S)). [0087] In one embodiment, VH CDR2 can be I-X5-YDG-X6X7X8 (SEQ ID NO: 52). In some embodiments, X5, X6, X7, or X8 can be Asparagine (N), Serine (S), Lysine (K), Histidine (H), Arginine (R), Glutamate (E)). In some embodiments, X5 or X8 can be a non-polar amino acid residue (e.g., phenylalanine (F), Leucine (L)). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [0088] In one embodiment, VH CDR3 can be AR- X12X13X14-MDV (SEQ ID NO: 53). In some embodiments, X12 or X13 can be a hydrophilic polar amino acid residue (e.g., Serine (S), Arginine (R)). In other embodiments, X13 or X14 can be a hydrophobic, non-polar amino acid residue (e.g., phenylalanine (F), glycine (G)). [0089] In one embodiment, VH CDR1 can be GFTF- X9X10X11-G (SEQ ID NO: 54). In some embodiments, X9 or X10 can be a hydrophilic polar amino acid residue (e.g., Asparagine (N), Serine (S), Threonine (T), Arginine (R)). In other embodiments, X11 can be a non-polar amino acid residue (e.g., phenylalanine (F), or tyrosine (Y)). [0090] In one embodiment, VL CDR1 can be SGSIATN- X15 (SEQ ID NO: 58). In some embodiments, X15 can be a non-polar amino acid residue (e.g., phenylalanine (F), or tyrosine (Y)). [0091] In embodiments, hydrophobic, non-polar amini residues include glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and tryptophan (W). Hydrophilic polar amino acid residues can include serine (S), arginine (R), threonine (T), cysteine (C), asparagine (N), glutamine (Q), lysine (K), histidine (H), aspartic acid (D), glutamic acid/glutamate (E), and tyrosine (Y). In embodiments, neutral amino acid residues include serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), tyrosine (Y), glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), arginine (R), and tryptophan (W). In embodiments, tyrosine (Y), tryptophan (W), lysine (K), and methionine (M) can be polar or nonpolar. [0092] As used herein, the term "epitope" can include any protein determinant capable of specific binding to an immunoglobulin, a scFv, or a T-cell receptor. The variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. For example, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three- dimensional antigen-binding site. This quaternary antibody structure forms the antigen- binding site at the end of each arm of the Y. Epitopic determinants can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- terminal or C-terminal peptides of a polypeptide. In some embodiments, the antibodies are directed to the EC domain of CD99. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In one embodiment, the antibodies can be directed to DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [0093] CD99 (SEQ ID NO: 32; GenBank Accession No.: CAG29282, 185 amino acids in length): 1 margaalall lfgllgvlva apDGGFDLSD ALPDNENKKP TAIPKKPSAG DDFDLGDAVV 61 DGENDDPRPP NPPKPMPNPN PNHPSSSGSF SDADLADGVS GGEGKGGSDG GGSHRKEGEE 121 ADAPGVIPGI VGAVVVAVAG AISSFIAYQK KKLCFKENAE QGEVDMESHR NANAEPAVQR 181 TLLEK [0094] The lower-case residues correspond to the signal sequence (SS); bolded residues correspond to the extracellular domain (EC); underlined residues correspond to the transmembrane domain (TD); and italicized residues correspond to the cytoplasmic domain (C). [0095] As used herein, the terms "immunological binding," and "immunological binding properties" can refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the equilibrium binding constant (KD) of the interaction, wherein a smaller KD represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen- binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). The ratio of Koff /Kon enables the cancellation of parameters not related to affinity and is equal to the equilibrium binding constant (KD). (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the invention can specifically bind to a CD99 epitope when the equilibrium binding constant (KD) is ≤10 μΜ, ≤ 10 nM, ≤ 10 pM, or ≤ 100 pM to about 1 pM, as measured by kinetic assays such as radioligand binding assays or similar assays known to those skilled in the art, such as BIAcore. [0096] “Specifically binds” or “has specificity to,” can refer to an antibody that binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it can bind to a random, unrelated epitope. For example, the CD99 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 antibody can be monovalent or bivalent, and comprises a single or double chain. Functionally, the binding affinity of the CD99 antibody is within the range of 10−5M to 10−12 M. For example, the binding affinity of the CD99 antibody is from 10−6 M to 10−12 M, from 10−7 M to 10−12 M, from 10−8 M to 10−12 M, from 10−9 M to 10−12 M, from 10−5 M to 10−11 M, from 10−6 M to 10−11 M, from 10−7 M to 10−11 M, from 10−8 M to 10−11 M, from 10−9 M to 10−11 M, from 10−10 M to 10−11 M, from 10−5 M to 10−10M, from 10−6 M to 10−10 M, from 10−7 M to 10−10 M, from 10−8 M to 10−10M, from 10−9 M to 10−10 M, from 10−5 M to 10−9 M, from 10−6 M to 10−9M, from 10−7 M to 10−9 M, from 10−8 M to 10−9 M, from 10−5 M to 10−8 M, from 10−6 M to 10−8 M, from 10−7 M to 10−8 M, from 10−5 M to 10−7 M, from 10−6 M to 10−7 M, or from 10−5 M to 10−6 M. [0097] A CD99 protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, can be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components. A CD99 protein or a derivative, fragment, analog, homolog, or ortholog thereof, coupled to a proteoliposome can be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components. [0098] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by ascertaining whether the former prevents the latter from binding to CD99. For example, if the human monoclonal antibody being tested competes with the human monoclonal antibody of the invention, as shown by a decrease in binding by the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same, or to a closely related, epitope. [0099] Another way to determine whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to pre-incubate the human monoclonal antibody of the invention with the CD99 protein, with which it is normally reactive, and then add the human monoclonal antibody being tested to determine if the human monoclonal antibody being tested is inhibited in its ability to bind CD99. If the human monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention. Screening of human monoclonal antibodies of the invention can be also carried out by utilizing CD99 and determining whether the test monoclonal antibody is able to neutralize CD99. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00100] Referring to FIG.12, ELISA results demonstrate competitive binding between PILR^ and anti-CD99, indicating a shared epitope on CD99. Accordingly, in certain embodiments, the anti-CD99 antibody binds an epitope that overlaps with that of PILR^ and/or anti-CD99. Accordingly, aspects of the invention are drawn to an an-CD99 antibody of the present disclosure, wherein the anti-CD99 antibody competes with binding to CD99 or binds the same epitope as PILR^ or an anti-PILRα antibody. [00101] A first binding protein (e.g., antibody or ligand) “binds to the same epitope” as a second binding protein (e.g., antibody or ligand) if the first binding protein binds to the same site on a target compound that the second binding protein binds, or binds to a site that overlaps (e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence or other molecular feature (e.g., glycosyl group, phosphate group, or sulfate group)) with the site that the second binding protein binds. [00102] A first binding protein (e.g., antibody or ligand) “competes for binding” with a second binding protein (e.g., antibody) if the binding of the first binding protein to its epitope decreases (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) the amount of the second binding protein that binds to its epitope. The competition can be direct (e.g., the first binding protein binds to an epitope that is the same as, or overlaps with, the epitope bound by the second binding protein), or indirect (e.g., the binding of the first binding protein to its epitope causes a steric change in the target compound that decreases the ability of the second binding protein to bind to its epitope). [00103] Various procedures known within the art can be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). [00104] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol.14, No.8 (April 17, 2000), pp.25-28). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00105] The term "monoclonal antibody" or “mAb” or "MAb" or "monoclonal antibody composition", as used herein, can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. The complementarity determining regions (CDRs) of the monoclonal antibody are identical in the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with an epitope of the antigen characterized by a unique binding affinity for it. [00106] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro. [00107] The immunizing agent can include the protein antigen, a fragment thereof or a fusion protein thereof. For example, peripheral blood lymphocytes can be used if cells of human origin are desired, or spleen cells or lymph node cells can be used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59- 103). Immortalized cell lines can be transformed mammalian cells, such as myeloma cells of rodent, bovine and human origin. For example, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells. [00108] Immortalized cell lines that are useful are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. For example, immortalized cell lines can be murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63)). [00109] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. For example, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen. [00110] After the hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal. [00111] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. [00112] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No.4,816,567 (incorporated herein by reference in its entirety). DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (See U.S. Patent No.4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 polypeptide can be substituted for the constant domains of an antibody of the invention or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody. [00113] Fully human antibodies, for example, are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. “Humanized antibodies” can be antibodies from non-human species whose light chain and heavy chain protein sequences have been modified to increase their similarity to antibody variants produced in humans. Humanized antibodies are antibody molecules derived from a non-human species antibody that bind the antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen-binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen-binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) For example, the non-human part of the antibody (such as the CDR(s) of a light chain and/or heavy chain) can bind to the target antigen. A humanized monoclonal antibody can also be referred to a “human monoclonal antibody” herein. [00114] Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain shuffling (U.S. Pat. No.5,565,332, which is incorporated by reference in its entirety). “Humanization” (also called Reshaping or CDR-grafting) is a well-established technique understood by the skilled artisan for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (i.e., rodent) and for improving their activation of the human immune system (See, for example, Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). "Humanization of an anti-CD34 monoclonal antibody DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 by complementarity-determining region grafting based on computer-assisted molecular modeling". J Biochem.144 (1): 115–20). [00115] Human monoclonal antibodies, such as fully human and humanized antibodies, can be prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al, 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be utilized and can be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). [00116] In addition, antibodies can also be produced using other techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.5,545,807; 5,545,806; 5,569,825; 5,625, 126; 5,633,425; 5,661,016, and in Marks et al, Bio/Technology 10, 779-783 (1992); Lonberg et al, Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol.1365-93 (1995). [00117] Human antibodies can additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication no. WO94/02602 and U.S. Patent No.6,673,986). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides the modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. A non-limiting example of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules. Thus, using such a technique, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol.73:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Creative BioLabs (Shirley, NY) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described herein. [00118] An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No.5,939,598. It can be obtained by a method, which includes deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker. [00119] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No.5,916,771. This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00120] In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication No. WO 99/53049. [00121] The antibody can be expressed by a vector containing a DNA segment encoding the single chain antibody described herein. Vectors include, but are not limited to, chemical conjugates such as described in WO 93/64701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618), which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage, viral vectors, etc. The vectors can be chromosomal, non-chromosomal or synthetic. Retroviral vectors can also be used, and include moloney murine leukemia viruses. DNA viral vectors can also be used, and include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (See Geller, A. I. et al, J. Neurochem, 64:487 (1995); Lim, F., et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al, Proc Natl. Acad. Sci.: U.S.A.90:7603 (1993); Geller, A. I., et al, Proc Natl. Acad. Sci USA 87: 1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al, Science, 259:988 (1993); Davidson, et al, Nat. Genet 3 :219 (1993); Yang, et al, J. Virol.69:2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M. G.. et al, Nat. Genet.8: 148 (1994). [00122] Pox viral vectors introduce the gene into the cell’s cytoplasm. Avipox virus vectors result in only a short-term expression of the nucleic acid. Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus (HSV) vectors can be used for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The vector chosen will depend upon the target cell and the condition being treated. The introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaP04 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors. [00123] The vector can be employed to target essentially any target cell. For example, stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and can be useful in delivering the vector to the target cell. (See Bobo et al, Proc. Natl. Acad. Sci. USA 91 :2076-2080 (1994); Morrison et al, Am. J. Physiol.266:292- 305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration. [00124] These vectors can be used to express large quantities of antibodies that can be used in a variety of ways, for example, to detect the presence of CD99 in a sample. The antibody can also be used to try to bind to and disrupt a CD99 activity. [00125] Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No.4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen can be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments. [00126] Heteroconjugate antibodies are also within the scope of the invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies can, for example, target immune system cells to unwanted cells (see U.S. Patent No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO 92/200373; EP 03089). The antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.4,676,980. [00127] The antibody of the invention can be modified with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC). (See Caron et al, J. Exp Med., 176: 1191-1195 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al, Anti-Cancer Drug Design, 3 : 219-230 (1989)). [00128] In certain embodiments, an antibody of the invention can comprise an Fc variant comprising an amino acid substitution which alters the antigen-independent effector functions of the antibody, in particular the circulating half-life of the antibody. Such antibodies exhibit increased or decreased binding to FcRn when compared to antibodies lacking these substitutions, therefore, have an increased or decreased half-life in serum, respectively. Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered antibody is desired, e.g., to treat a chronic disease or disorder. In contrast, Fc variants with decreased FcRn binding affinity have shorter halt-lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time can be advantageous, e.g., for in vivo diagnostic imaging or in situations where the starting antibody has toxic side effects when present in the circulation for prolonged periods. Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity can be desired include those applications in which localization to the brain, kidney, and/or liver is desired. In one embodiment, the Fc-variant containing antibodies can exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the altered antibodies of the invention exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space. In one embodiment, an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the "FcRn binding loop" of an Fc domain. The FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in PCT Publication No. WO 05/047327 which is incorporated by reference herein. In certain exemplary embodiments, the antibodies, or fragments thereof, of the invention comprise an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E and S304D (EU numbering). [00129] In some embodiments, mutations are introduced to the constant regions of the mAb such that the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the mAb is altered. For example, the mutation is a LALA mutation in the CH2 domain. In one embodiment, the antibody (e.g., a human antibody or bispecific antibody (bsAb)) contains DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 mutations on one scFv unit of the heterodimeric mAb, which reduces the ADCC activity. In another embodiment, the mAb contains mutations on both chains of the heterodimeric mAb, which completely ablates the ADCC activity. For example, the mutations introduced one or both scFv units of the mAb are LALA mutations in the CH2 domain. These mAbs with variable ADCC activity can be optimized such that the mAbs exhibits maximal selective killing towards cells that express one antigen that is recognized by the mAb, however exhibits minimal killing towards the second antigen that is recognized by the mAb. [00130] In other embodiments, antibodies of the invention for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG1 or IgG4 heavy chain constant region, which can be altered to reduce or eliminate glycosylation. For example, an antibody of the invention can also comprise an Fc variant comprising an amino acid substitution which alters the glycosylation of the antibody. For example, the Fc variant can have reduced glycosylation (e.g., N- or O-linked glycosylation). In some embodiments, the Fc variant comprises reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS. In a partic ular embodiment, the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering). In embodiments, the antibody comprises an IgGl or IgG4 constant region comprising an S228P and a. T299A mutation (EU numbering). [00131] Exemplary amino acid substitutions which confer reduced or altered glycosylation are disclosed in PCT Publication No. WO05/018572, which is incorporated by reference herein in its entirety. In some embodiments, the antibodies of the invention, or fragments thereof, are modified to eliminate glycosylation. Such antibodies, or fragments thereof, can be referred to as "agly" antibodies, or fragments thereof, (e.g. "agly" antibodies). While not wishing to be bound by theory, "agly" antibodies, or fragments thereof, can have an improved safety and stability profile in vivo. Exemplary agly antibodies, or fragments thereof, comprise an aglycosylated Fc region of an IgG4 antibody which is devoid of Fc-effector function thereby eliminating the potential for Fc mediated toxicity to the normal vital tissues and cells that express CD99. In yet other embodiments, antibodies of the invention, or fragments thereof, comprise an altered glycan. For example, the antibody can have a reduced number of fucose residues on an N-glycan at Asn297 of the Fc region, i.e., is afucosylated. In another embodiment, the antibody can have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00132] The invention also is directed to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). [00133] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Non-limiting examples include 212Bi, 131I, 131In, 90Y, and 186Re. [00134] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See PCT Publication No. WO94/11026 and U.S. Patent No.5,736,137). [00135] Those of ordinary skill in the art understand that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the invention. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference). [00136] Coupling can be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In one embodiment, binding is covalent binding. Covalent binding can be achieved by direct condensation of existing side DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun.133 : 1335- 2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al, Science 238: 1098 (1987)). Non-limiting examples of linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res.44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Non-limiting examples of useful linkers that can be used with the antibodies of the invention include: (i) EDC (l-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4- succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2- pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC- SPDP (sulfosuccinimidyl 6 [3-(2- pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo- NHS (-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC. [00137] The linkers described herein contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo- NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone. [00138] The antibodies disclosed herein can also be formulated as immunoliposomes. [00139] Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos.4,485,045 and 4,544,545. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00140] Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. [00141] Non-limiting examples of useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al, J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. [00142] Bi-specific Antibodies [00143] Unlike a monospecific antibody, which recognizes a single antigen or type of antigen, a bi-specific antibody (bsAb) is an antibody comprising two variable domains or scFv units such that the resulting antibody recognizes two different antigens. The present invention provides for bi-specific antibodies that recognize CD99 and a second antigen. An antibody or antigen-binding fragment specific to CD99 can be combined with a second antigen-binding fragment specific to an immune cell to generate a bispecific antibody. In some embodiments, the immune cell is selected from the group consisting of a T cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a phagocyte, a natural killer cell, an eosinophil, a basophil, and a mast cell. Molecules on the immune cell which can be targeted include, for example, CD3, CD16, CD19, CD28, and CD64. Other non-limiting examples include PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer-cell immunoglobulin-like receptors (KIRs), and CD47. Exemplary second antigens include tumor associated antigens (e.g., LINGO1, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, Mucins, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin), cytokines (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF- α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL), and cell surface receptors (e.g. IGF1R and/or PILRa). A bi-specific antibody of the present invention comprises a heavy chain and a light chain combination or scFv of the CD99 antibodies disclosed herein. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00144] Sequence information related to human LINGO1 is accessible in public databases by GenBank Accession numbers NP_001288115.1 (protein) and NM_001301186.1 (nucleic acid). [00145] The genomic sequence for human IGF1R has GenBank Accession No. NG_009492.1. Sequence information related to human IGF1R is accessible in public databases by GenBank Accession numbers NP_000866.1 (protein) and NM_000875.5 (nucleic acid). [00146] Bi-specific antibodies of the present invention can be constructed using methods known in the art. In some embodiments, the bi-specific antibody is a single polypeptide wherein the two scFv fragments are joined by a long linker polypeptide, of sufficient length to allow intramolecular association between the two scFv units to form an antibody. In other embodiments, the bi-specific antibody is more than one polypeptide linked by covalent or non-covalent bonds. [00147] In another embodiment, the bi-specific antibody is constructed using the "knob into hole" method (Ridgway et al, Protein Eng 7:617-621 (1996)). In this method, the Ig heavy chains of the two different variable domains are reduced to selectively break the heavy chain pairing while retaining the heavy-light chain pairing. The two heavy-light chain heterodimers that recognize two different antigens are mixed to promote heteroligation pairing, which is mediated through the engineered "knob into holes" of the CH3 domains. [00148] In another embodiment, the bi-specific antibody can be constructed through exchange of heavy-light chain dimers from two or more different antibodies to generate a hybrid antibody where the first heavy-light chain dimer recognizes CD99 and the second heavy-light chain dimer recognizes a second antigen. In some embodiments, the bi-specific antibody can be constructed through exchange of heavy-light chain dimers from two or more different antibodies to generate a hybrid antibody where the first heavy-light chain dimer recognizes a second antigen and the second heavy-light chain dimer recognizes CD99.The mechanism for heavy-light chain dimer is similar to the formation of human IgG4, which also functions as a bispecific molecule. Dimerization of IgG heavy chains is driven by intramolecular force, such as the pairing the CH3 domain of each heavy chain and disulfide bridges. Presence of a specific amino acid in the CH3 domain (R409) has been shown to promote dimer exchange and construction of the IgG4 molecules. Heavy chain pairing is also stabilized further by interheavy chain disulfide bridges in the hinge region of the antibody. Specifically, in IgG4, the hinge region contains the amino acid sequence Cys-Pro-Ser-Cys (in comparison to the stable IgGl hinge region which contains the sequence Cys-Pro-Pro-Cys) at DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 amino acids 226- 230. This sequence difference of Serine at position 229 has been linked to the tendency of IgG4 to form intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al, 2007, Science 317: 1554-1557 and Labrijn, A.F. et al, 2011, Journal of Immunol 187:3238-3246). [00149] Therefore, bi-specific antibodies of the present invention can be created through introduction of the R409 residue in the CH3 domain and the Cys-Pro-Ser-Cys sequence in the hinge region of antibodies that recognize CD99 or a second antigen, so that the heavy-light chain dimers exchange to produce an antibody molecule with one heavy-light chain dimer recognizing CD99 and the second heavy-light chain dimer recognizing a second antigen, wherein the second antigen is any antigen disclosed herein. Known IgG4 molecules can also be altered such that the heavy and light chains recognize CD99 or a second antigen, as disclosed herein. Use of this method for constructing the bi-specific antibodies of the present invention can be beneficial due to the intrinsic characteristic of IgG4 molecules wherein the Fc region differs from other IgG subtypes in that it interacts poorly with effector systems of the immune response, such as complement and Fc receptors expressed by certain white blood cells. This specific property makes these IgG4-based bi-specific antibodies attractive for therapeutic applications, in which the antibody is required to bind the target(s) and functionally alter the signaling pathways associated with the target(s), however not trigger effector activities. [00150] In some embodiments, mutations are introduced to the constant regions of the bsAb such that the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb is altered. For example, the mutation is an LALA mutation in the CH2 domain. In one aspect, the bsAb contains mutations on one scFv unit of the heterodimeric bsAb, which reduces the ADCC activity. In another aspect, the bsAb contains mutations on both chains of the heterodimeric bsAb, which completely ablates the ADCC activity. For example, the mutations introduced one or both scFv units of the bsAb are LALA mutations in the CH2 domain. These bsAbs with variable ADCC activity can be optimized such that the bsAbs exhibits maximal selective killing towards cells that express one antigen that is recognized by the bsAb, however exhibits minimal killing towards the second antigen that is recognized by the bsAb. [00151] The bi-specific antibodies disclosed herein can be useful in treatment of diseases or medical conditions, for example, cancer. [00152] [00153] Use of Antibodies Against CD99 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00154] Antibodies specifically binding a CD99 protein or a fragment thereof of the invention can be administered for the treatment a CD99 associated disease or disorder. A "CD99-associated disease or disorder" includes disease states and/or symptoms associated with a disease state, where increased levels of CD99 and/or activation of cellular signaling pathways involving CD99 are found. Exemplary CD99-associated diseases or disorders include, but are not limited to, cancer and conditions for which inflammation or auto- immunity are inherent to the pathophysiology. [00155] Many cancers overexpress CD99 and the upregulation of CD99 is associated with high risk prognostic factors. CD99 is a cell surface protein with unique features and only partly defined mechanisms of action. CD99 is involved in biological processes such as cell adhesion, migration, death, differentiation and diapedesis, and it influences processes associated with inflammation, immune responses and cancer. CD99 is frequently overexpressed in many types of tumors, such as pediatric tumors including Ewing sarcoma and specific subtypes of leukemia, as well as synovial sarcoma, malignant peripheral nerve sheath tumors, astrocytoma/glioblastoma, pancreatic endocrine tumors, GI/pulmonary neuroendocrine tumors, prostate cancer, acute lymphoblastic leukemia, acute myeloid leukemia, stem cells in myelodysplastic syndrome, and lymphomas. [00156] Antibodies of the invention, including bi-specific, polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents. Such agents will be employed to treat or prevent cancer in a subject, increase vaccine efficiency or augment a natural immune response. An antibody preparation, for example, one having high specificity and high affinity for its target antigen, is administered to the subject and will have an effect due to its binding with the target. Administration of the antibody can abrogate or inhibit or interfere with an activity of the CD99 protein. [00157] A specific dosage and treatment regimen for any patient will depend upon a variety of factors, including the antibodies, variant or derivative thereof used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be 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 by pharmacological and pharmacokinetic principles well known in the art. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00158] Antibodies of the invention specifically binding a CD99 protein or fragment thereof can be administered for the treatment of a cancer in the form of pharmaceutical compositions. Principles and considerations involved in preparing therapeutic pharmaceutical compositions comprising the antibody, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 20th ed. (Alfonso R. Gennaro, et al, editors) Mack Pub. Co., Easton, Pa., 2000; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol.4), 1991, M. Dekker, New York. [00159] A therapeutically effective amount of an antibody of the invention can be the amount needed to achieve a therapeutic objective. As noted herein, this can be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. The dosage administered to a subject (e.g., a patient) of the antigen- binding polypeptides described herein can be 0.1 mg/kg to 100 mg/kg of the patient's body weight, between 0.1 mg/kg and 20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of the patient's body weight. Human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the disclosure can be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention can be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies can range, for example, from twice daily to once a week. [00160] Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 one active compound as necessary for the indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine (e.g. IL-15), chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. [00161] The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. [00162] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. [00163] Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. [00164] An antibody according to the invention can be used as an agent for detecting the presence of CD99 (or a protein fragment thereof) in a sample. For example, the antibody can contain a detectable label. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) can be used. The term "labeled", with regard to the probe or antibody, can encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently- labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 detected with fluorescently-labeled streptavidin. The term "biological sample" can include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA includes Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. [00165] Procedures for conducting immunoassays are described, for example in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol.42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. [00166] Antibodies directed against a CD99 protein (or a fragment thereof) can be used in methods known within the art relating to the localization and/or quantitation of a CD99 protein (e.g., for use in measuring levels of the CD99 protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific to a CD99 protein, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to herein as "therapeutics"). [00167] An antibody specific for a CD99 protein of the invention can be used to isolate a CD99 polypeptide by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Antibodies directed against a CD99 protein (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. [00168] Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 bioluminescent materials, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S, 32P or 3H. [00169] The antibodies or agents of the invention (also referred to herein as "active compounds"), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such pharmaceutical compositions can comprise the antibody or agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Non-limiting examples of such carriers or diluents include, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. [00170] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, transarterial (e.g., injection), intratumoral (e.g., injection), oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [00171] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In embodiments, the composition is sterile and is fluid to the extent that easy syringeability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. [00172] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. For example, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional ingredient from a previously sterile-filtered solution thereof. [00173] Oral compositions can include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. [00174] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. [00175] Systemic administration can also be by transmucosal or transdermal means. [00176] For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as known in the art. [00177] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. [00178] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.4,522,811. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00179] Oral or parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. [00180] One exemplary embodiment includes an antibody composition that comprises at least one antibody. The antibody can comprise two heavy chains and two light chains. In embodiments, the heavy chain CDRs are identical to reference germline CDRs found between residues 26 and 33, residues 51 and 58, and residues 97 and 113 according to IMGT numbering of SEQ ID NO: 29. In an alternate embodiments, the heavy chain CDRs are identical to reference germline CDRs found between residues 97 and 108 according to IMGT numbering of SEQ ID NO: 33. In other embodiments, the heavy chain CDRs can be identical to reference germline CDRs found between residues 97 and 110 according to IMGT numbering of SEQ ID NO: 34. In embodiments, at least one of the heavy chain CDRs differs by a single amino acid substitution relative to its reference CDR. For example, the heavy chain CDRs include one or more of the following residue substitutions: 30Pro, 32His of SEQ ID NO: 29 relative to SEQ ID NO: 40. For example, the heavy chain CDRs include one or more of the following residue substitutions: 31Asn, 103Gly of SEQ ID NO: 33 relative to SEQ ID NO: 40. For example, the heavy chain CDRs include one or more of the following residue substitutions: 30 or 31Ser; 31or 32Lys of SEQ ID NO: 34 relative to SEQ ID NO: 40. In an embodiment, the light chain CDRs are identical to reference germline CDRs found between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:30. In another embodiment, the light chain CDRs can be identical to reference germline CDRs found between residues 26 and 34, residues 52 and 58, and residues 97 and 104 according to IMGT numbering of SEQ ID NO: 110. In certain embodiments, at least one of the light chain CDRs differs by a single amino acid substitution relative to its reference CDR. For example, the light chain CDRs include one or more of the following residue substitutions: 29Asn; 30Thr; or 51Asn of SEQ ID NO: 30 relative to SEQ ID NO: 41. For example, the light chain CDRs include one or more of the following residue substitutions: 26Ala; 28Pro; 29Lys; or 30Gln of SEQ ID NO: 110 relative to SEQ ID NO: 41. For example, the light chain CDRs include one or more of the following residue DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 substitutions: 57Asn; 100His; 102Ser; or 104Trp of SEQ ID NO: 31 relative to SEQ ID NO: 42. In an embodiment, the antibody binds to an epitope that comprises amino residues at positions 23 to 123 of the extracellular domain of CD99 comprising SEQ ID NO: 32. [00181] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The pharmaceutical compositions can be incorporated into a kit suitable for therapeutic administration of the pharmaceutical compositions. In embodiments, the kit includes an applicator to administer the pharmaceutical composition. In embodiments, the kit includes a syringe, a needle, or other applicator for administration of at least one antibody fragment (as disclosed herein) to a subject. The kit can further include instructions for use. [00182] Methods of Treatment [00183] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can refer to prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. [00184] The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a cancer, or other cell proliferation-related diseases or disorders. Such diseases or disorders include but are not limited to, e.g., those diseases or disorders associated with aberrant expression of CD99. For example, the methods are used to treat, prevent or alleviate a symptom cancer. In an embodiment, the methods are used to treat, prevent or alleviate a symptom of a solid tumor such as a sarcoma (e.g., Ewing sarcoma or synovial sarcoma). Non-limiting examples of other tumors that can be treated by embodiments herein comprise lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer , malignant peripheral nerve sheath tumor, astrocytoma, glioblastoma, pancreatic endocrine tumor, GI/pulmonary neuroendocrine tumor, acute lymphoblastic leukemia, acute myeloid leukemia, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 myelodysplastic syndrome, lymphoma , stomach cancer, or a combination thereof. Additionally, the methods of the invention can be used to treat hematologic cancers such as leukemia and lymphoma. Alternatively, the methods can be used to treat, prevent or alleviate a symptom of a cancer that has metastasized. [00185] Accordingly, in one aspect, the invention provides methods for preventing, treating or alleviating a symptom cancer or a cell proliferative disease or disorder in a subject by administering to the subject a monoclonal antibody, scFv antibody or bi- specific antibody of the invention. For example, an anti-CD99 antibody can be administered in therapeutically effective amounts. [00186] Subjects at risk for cancer or cell proliferation-related diseases or disorders can include patients who have a family history of cancer or a subject exposed to a known or suspected cancer-causing agent. Administration of a prophylactic agent can occur prior to the manifestation of cancer such that the disease is prevented or, alternatively, delayed in its progression. [00187] In another aspect, tumor cell growth is inhibited by contacting a cell with an anti- CD99 antibody of the invention. The cell can be any cell that expresses CD99. [00188] Also included in the invention are methods of increasing or enhancing an immune response to an antigen. An immune response is increased or enhanced by administering to the subject a monoclonal antibody, scFv antibody, or bi-specific antibody of the invention. The immune response is augmented for example by augmenting antigen specific T effector function. The antigen is a viral (e.g. HIV), bacterial, parasitic or tumor antigen. The immune response is a natural immune response. By natural immune response is meant an immune response that is a result of an infection. The infection is a chronic infection. Increasing or enhancing an immune response to an antigen can be measured by a number of methods known in the art. For example, an immune response can be measured by measuring any one of the following: T cell activity, T cell proliferation, T cell activation, production of effector cytokines, and T cell transcriptional profile. [00189] Alternatively, the immune response is a response induced due to a vaccination. [00190] Accordingly, in another aspect the invention provides a method of increasing vaccine efficiency by administering to the subject a monoclonal antibody or scFv antibody of the invention and a vaccine. The antibody and the vaccine are administered sequentially or concurrently. The vaccine is a tumor vaccine a bacterial vaccine or a viral vaccine. [00191] Chimeric antigen receptor (CAR) T-cell therapies DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00192] Cellular therapies, such as chimeric antigen receptor (CAR) T-cell therapies expressing an anti-CD99 antibody as described herein, are also provided. CAR T-cell therapies redirect a patient’s T-cells to kill tumor cells by the exogenous expression of a CAR on a T-cell, for example. A CAR can be a membrane spanning fusion protein that links the antigen recognition domain of an antibody to the intracellular signaling domains of the T-cell receptor and co-receptor. A suitable cell can be used, for example, that can secrete an anti- CD99 antibody of the present invention (or alternatively engineered to express an anti-CD99 antibody as described herein to be secreted). The anti-CD99 “payloads” to be secreted, can be, for example, minibodies, ScFvs, IgG molecules, bispecific fusion molecules, and other antibody fragments as described herein. Upon contact or engineering, the cell described herein can then be introduced to a patient in need of a treatment by infusion therapies known to one of skill in the art. The patient can have a CD99-associated disease, such as the cancers described herein. The cell (e.g., a T cell) can be, for instance, T lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination thereof, without limitation. Exemplary CARs and CAR factories useful in aspects of the invention include those disclosed in, for example, PCT/US2015/067225 and PCT/US2019/022272, each of which are hereby incorporated by reference in their entireties. In one embodiment, the CD99 antibodies discussed herein can be used in the construction of multi-specific antibodies or as the payload for a CAR-T cell. [00193] Accordingly, aspects of the invention can be drawn to a genetically engineered cell comprising a chimeric antigen receptor, wherein the genetically engineered cell expresses and/or secretes an anti-CD99 antibody as described herein. [00194] [00195] Combinatory Methods [00196] Compositions of the invention as described herein can be administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the compositions described herein include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide). [00197] In additional embodiments, the compositions of the invention as described herein can be administered in combination with cytokines. Cytokines that can be administered with the compositions 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-α. [00198] In additional embodiments, the compositions described herein can be administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy. [00199] In some embodiments, the compositions described herein can be administered in combination with other immunotherapeutic agents. Non-limiting examples of immunotherapeutic agents include simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, and 3F8. [00200] The invention provides for methods of treating cancer in a patient by administering two antibodies that bind to the same epitope of the CD99 protein or, alternatively, two different epitopes of the CD99 protein. Alternatively, the cancer is treated by administering a first antibody that binds to CD99 and a second antibody that binds to a protein other than DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 CD99. For example, the other protein other than CD99 can include, but is not limited to, LIGO1, IGF-1R, and PILRa. For example, the other protein other than CD99 is a tumor- associated antigen. [00201] In some embodiments, the invention provides administration of an anti-CD99 antibody alone or with an additional antibody that recognizes another protein other than CD99, with cells that are capable of effecting or augmenting an immune response. For example, these cells can be peripheral blood mononuclear cells (PBMC), or any cell type that is found in PBMC, e.g., cytotoxic T cells, macrophages, and natural killer (NK) cells. [00202] Additionally, the invention provides administration of an antibody that binds to the CD99 protein and an anti-neoplastic agent, such a small molecule, a growth factor, a cytokine or other therapeutics including biomolecules such as peptides, peptidomimetics, peptoids, polynucleotides, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, and proteases. Small molecules include, but are not limited to, inorganic molecules and small organic molecules. Suitable growth factors or cytokines include an IL- 2, GM-CSF, IL-12, and TNF-alpha, as well as checkpoint inhibition polypeptides known in the art, or macrophage dysfunction polypeptides (such as CSF1R). Small molecule libraries are known in the art. (See, Lam, Anticancer Drug Des., 12: 145, 1997.) Poly nucleotides include RNA, DNA, and non-coding RNAs. In embodiments, polynucleotides include, but are not limited to small interfering (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), piwi- interacting RNA (piRNA), small nucleolar RNA (snoRNA), and long noncoding RNA (lncRNA). [00203] Diagnostic Assays [00204] The anti-CD99 antibodies can be used diagnostically to, for example, monitor the development or progression of cancer (e.g., Ewing sarcoma) as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. [00205] In some aspects, for diagnostic purposes the anti-CD99 antibody of the invention is linked to a detectable moiety, for example, so as to provide a method for detecting a cancer cell in a subject at risk of or suffering from a cancer. [00206] The detectable moieties can be conjugated directly to the antibodies or fragments, or indirectly by using, for example, a fluorescent secondary antibody. Direct conjugation can be accomplished by standard chemical coupling of, for example, a fluorophore to the antibody or antibody fragment, or through genetic engineering. Chimeras, or fusion proteins can be constructed which contain an antibody or antibody fragment coupled to a fluorescent DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 or bioluminescent protein. For example, Casadei, et al, describe a method of making a vector construct capable of expressing a fusion protein of aequorin and an antibody gene in mammalian cells. [00207] As used herein, the term "labeled", with regard to the probe or antibody, can encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject (such as a biopsy), as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect cells that express CD99 in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of CD99 include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. Furthermore, in vivo techniques for detection of CD99 include introducing into a subject a labeled anti-CD99 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In the case of "targeted" conjugates, that is, conjugates which contain a targeting moiety— a molecule or feature designed to localize the conjugate within a subject or animal at site or sites, localization can refer to a state when an equilibrium between bound, "localized", and unbound, "free" entities within a subject has been essentially achieved. The rate at which such equilibrium is achieved depends upon the route of administration. For example, a conjugate administered by intravenous injection can achieve localization within minutes of injection. On the other hand, a conjugate administered orally can take hours to achieve localization. Alternatively, localization can simply refer to the location of the entity within the subject or animal at selected time periods after the entity is administered. By way of another example, localization is achieved when an moiety becomes distributed following administration. [00208] A reasonable estimate of the time to achieve localization can be made by one skilled in the art. Furthermore, the state of localization as a function of time can be followed by imaging the detectable moiety (e.g., a light-emitting conjugate) according to the methods of the invention, such as with a photodetector device. The "photodetector device" used can DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 have a high enough sensitivity to enable the imaging of faint light from within a mammal in a reasonable amount of time, and to use the signal from such a device to construct an image. [00209] In cases where it is possible to use light-generating moieties which are extremely bright, and/or to detect light-generating fusion proteins localized near the surface of the subject or animal being imaged, a pair of "night- vision" goggles or a standard high- sensitivity video camera, such as a Silicon Intensified Tube (SIT) camera (e.g., from Hammamatsu Photonic Systems, Bridgewater, N.J.), can be used. However, a more sensitive method of light detection is required. [00210] In extremely low light levels the photon flux per unit area becomes so low that the scene being imaged no longer appears continuous. Instead, it is represented by individual photons which are both temporally and spatially distinct form one another. Viewed on a monitor, such an image appears as scintillating points of light, each representing a single detected photon. By accumulating these detected photons in a digital image processor over time, an image can be acquired and constructed. In contrast to conventional cameras where the signal at each image point is assigned an intensity value, in photon counting imaging the amplitude of the signal carries no significance. The objective is to simply detect the presence of a signal (photon) and to count the occurrence of the signal with respect to its position over time. [00211] At least two types of photodetector devices, described herein, can detect individual photons and generate a signal which can be analyzed by an image processor. Reduced-Noise Photodetection devices achieve sensitivity by reducing the background noise in the photon detector, as opposed to amplifying the photon signal. Noise is reduced primarily by cooling the detector array. The devices include charge coupled device (CCD) cameras referred to as "backthinned", cooled CCD cameras. In the more sensitive instruments, the cooling is achieved using, for example, liquid nitrogen, which brings the temperature of the CCD array to approximately -120°C. "Backthinned" refers to an ultra- thin backplate that reduces the path length that a photon follows to be detected, thereby increasing the quantum efficiency. A sensitive backthinned cryogenic CCD camera is the "TECH 512", a series 200 camera available from Photometries, Ltd. (Tucson, Ariz.). [00120] "Photon amplification devices" amplify photons before they hit the detection screen. This class includes CCD cameras with intensifiers, such as microchannel intensifiers. A microchannel intensifier can contain a metal array of channels perpendicular to and co-extensive with the detection screen of the camera. The microchannel array is placed between the sample, subject, or animal to be imaged, and the camera. Most of the photons entering the channels of the array contact a side of a channel DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 before exiting. A voltage applied across the array results in the release of many electrons from each photon collision. The electrons from such a collision exit their channel of origin in a "shotgun" pattern and are detected by the camera. [00212] Even greater sensitivity can be achieved by placing intensifying microchannel arrays in series, so that electrons generated in the first stage in turn result in an amplified signal of electrons at the second stage. Increases in sensitivity, however, are achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel intensifier-based single-photon detection device is the C2400 series, available from Hamamatsu. [00213] Image processors process signals generated by photodetector devices which count photons in order to construct an image which can be, for example, displayed on a monitor or printed on a video printer. Such image processors can be sold as part of systems which include the sensitive photon-counting cameras described herein, and accordingly, are available from the same sources. The image processors can be connected to a personal computer, such as an IBM-compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif), which can be included as part of a purchased imaging system. Once the images are in the form of digital files, they can be manipulated by a variety of image processing programs (such as "ADOBE PHOTOSHOP", Adobe Systems, Adobe Systems, Mt. View, Calif.) and printed. [00214] In an embodiment, the biological sample contains protein molecules from the test subject. One preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. [00215] The invention also encompasses kits for detecting the presence of CD99 or a CD99-expressing cell in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting a cancer or tumor cell (e.g., an anti-CD99 scFv or monoclonal antibody) in a biological sample; means for determining the amount of CD99 in the sample; and means for comparing the amount of CD99 in the sample with a standard. The standard is, in some embodiments, a non-cancer cell or cell extract thereof. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect cancer in a sample. [00216] Other Embodiments [00217] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. [00218] [00219] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 EXAMPLES [00220] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results. EXAMPLE 1 [00221] Human Anti-CD99 Monoclonal Antibodies [00222] The treatment for pediatric solid tumor patients traditionally consists of a combination of chemotherapy, surgery, and radiation therapy. While these modalities are successful in curing patients with localized disease they are associated with substantial acute and long-term toxicities. Patients with widely metastatic or recurrent disease are rarely cured. Targeted therapies, designed specifically for individual tumors, allow a more focused approach to treatment aimed at improving overall survival and diminishing side effects. One therapeutic target with applicability to cancers such as sarcoma, prostate cancer, leukemias and lymphomas is a protein called CD99. [00223] Ewing sarcoma is a solid tumor arising from the bone and soft tissues affecting approximately 250 children, adolescents, and young adults each year. It is a model tumor for study given nearly universal membranous CD99 expression, disparate outcomes for patients with localized versus metastatic disease, and the unmet need for more effective therapies. Previous work demonstrated the ability of a 64Cu-labeled anti-CD99 monoclonal antibody (mAb) to detect micro-metastatic lesions in a Ewing sarcoma mouse model with high sensitivity and specificity. Antibody-engagement of CD99 has been shown to provoke Ewing cell aggregation and caspase-independent apoptosis. The ability of an anti-CD99 Ab to bind with specificity and induce cell death identify CD99 as a therapeutic target. [00224] Aspects of the invention are directed towards unique human anti-CD99 antibody clones derived from human-Ab phagemid library panning against a soluble CD99 protein. The antibodies generated from this endeavor bind with great affinity to soluble CD99 and patient-derived Ewing sarcoma cell lines. In vitro, they induce Ewing cell death in the presence of human effector cells and serum complement. In vivo binding of these antibody clones to Ewing sarcoma xenograft tumors, mechanisms of Ewing cell death following CD99 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 engagement, on-target off-tumor toxicity, and an exploration of the Ewing sarcoma human immune milieu will be conducted. EXAMPLE 2 [00225] Introduction [00226] Targeted therapies have played an increasingly important role in modern clinical oncology as a means by which to enhance treatment specificity and efficacy while diminishing toxicity. One potential target with applicability to sarcoma, prostate cancer, leukemias and lymphomas is CD99, though additional efforts are needed to optimize CD99- targeted treatment strategies. [00227] Ewing sarcoma will be investigated as a model system to study CD99 targeting. Ewing Sarcoma is a disease with nearly universal membranous CD99 expression, disparate outcomes for patients with localized versus metastatic disease, and an unmet need for more effective therapies. Previous work demonstrated the ability of a 64Cu-labeled anti-CD99 monoclonal antibody (mAb) to detect micro-metastatic lesions in a Ewing sarcoma mouse model with high sensitivity and specificity.1 Antibody-engagement of CD99 has been shown to provoke Ewing cell aggregation and caspase-independent apoptosis.2-4 Thus, the ability of an anti-CD99 Ab to bind with specificity and induce cell death identifies CD99 as a therapeutic target. Studies have led to the discovery of unique human anti-CD99 mAb clones derived from human-Ab phagemid library panning against a soluble CD99 protein. The top antibodies generated from these experiments bind with great affinity to soluble CD99 and patient-derived Ewing sarcoma cell lines. In vitro, they induce Ewing cell death in the presence of human effector cells and serum complement. Described herein are studies to analyze their in vivo activity in a humanized mouse model supporting next steps needed for translation of a human anti-CD99 mAb to the clinic. These goals will be accomplished through the following Aims: [00228] Aim 1: To define CD99 mAb activity in an Ewing sarcoma humanized mouse model. Without wishing to be bound by theory, a Ewing sarcoma humanized mouse model will allow a unique assessment of the Ewing tumor immune milieu, in vivo anti-CD99 mAb induced tumor cytotoxicity, and on-target off-tumor binding so as to further inform the safety and efficacy of an immunotherapeutic human anti-CD99 mAb. [00229] Aim 1(a)To examine the Ewing sarcoma tumor immune environment in a humanized Ewing sarcoma xenograft model pre- and post-treatment with human anti-CD99 mAb. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00230] Aim 1(b)To determine the effect of treatment with human anti-CD99 mAb on tumor cell growth. [00231] Aim 1(c)To investigate on-target off-tumor binding of human anti-CD99 mAb. [00232] Significance [00233] Ewing sarcoma affects approximately 250 children, adolescents, and young adults each year in the United States.5,6 Patients with chemotherapy-responsive, localized disease have an excellent 5-year overall survival of greater than 75%.7 Conversely, the presence of metastatic disease confers a very poor prognosis; less than 20% of these patients are cured.8,9 Approximately 20% of patients have metastatic disease at diagnosis on the basis of current imaging techniques; however, it is assumed that greater than 80% of newly diagnosed patients have micrometastatic disease.9 [00234] The mainstay of therapy for Ewing sarcoma consists of systemic chemotherapy, surgery and/or radiotherapy. Chemotherapy, while effective, can confer the risk of long-term cardiotoxicity, bone marrow injury, and infertility while surgery can be disfiguring or impact quality of life.10 Metastatic sites of disease, a tumor non-amenable to surgical resection, or a tumor with positive margins following resection necessitate the addition of radiotherapy.11,12 Radiation therapy, while highly effective, can cause significant damage to healthy tissues as well as the lifelong risk for secondary malignancy.13 For the subset of patients with metastatic, recurrent, or chemotherapy refractory disease, the above-mentioned modalities offer transient success and little improvement has been made in overall survival for decades.14 [00235] Ewing sarcoma is characterized histologically by small, round blue cells and a diffuse membranous immunohistochemical stain for CD99 (or MIC2), a 32kDa type I membrane glycoprotein.P9-P13 Ewing sarcoma is suited for a targeted approach to therapy given diffuse membranous immunohistochemical staining for CD99 (or MIC2). CD99 is a 32kDa type I membrane glycoprotein highly expressed in 100% of Ewing sarcoma specimens and believed to play a role in the differentiation and malignant phenotype of Ewing cells.4,15- 19 CD99 is biologically linked to EWS:FLI1, the pathognomonic ETS-family translocation present in up to 85% of Ewing tumors.20 Ectopic expression of the EWS:FLI1 translocation in mesenchymal stem cells can induce Ewing cell morphology and CD99 expression.21 For example, eighty-five percent or more of Ewing sarcoma cases harbor a chimeric fusion between the RNA binding protein EWS and an ETS family transcription factor (i.e., EWS:FLI1) creating a fusion oncogene (t(11;22)(q24;q12)) deemed relevant to tumor pathogenesis.P14,P15 In addition, the Ewing immune landscape has traditionally been described DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 as a “cold” immune state. However, the presence of CD68+ tumor-associated macrophages (TAMs) has been linked to poor clinical outcomes.P18-P20 [00236] In the absence of available EWS:FLI1 targeted agents, therapeutic antibodies targeting CD99 have been investigated. Anti-CD99 antibody blockade has been shown to induce Ewing cell aggregation and caspase-independent apoptosisP11, trigger Ewing cell death in p53 wild-type patient-derived cell linesP21, and more recently incite cell death by a process termed methuosis involving the IGF-1R/Ras/Rac1 signaling pathway. P22 However, the role of the human immune system in anti-CD99 mediated Ewing cell death has not yet been explored. [00237] Antibody-engagement of CD99 provokes Ewing cell aggregation and caspase- independent apoptosis.4 A human anti-CD99 single-chain variable fragment (scFv) with preferential binding to CD99 on Ewing sarcoma cell lines and tumors has been designed.2 Binding of CD99 by this scFv triggers cell death in p53 wild-type patient-derived cell lines, demonstrating downstream transcriptional modulation involving the ERK/MAPK pathway, mitochondrial dysfunction, and apoptosis signaling.3 This data indicates the need for a more in-depth examination of the mechanisms of Ewing cell death following CD99 antibody binding. [00238] As described herein, unique human anti-CD99 scFv’s derived from human-Ab phagemid library panning have been identified that that demonstrate in vitro mAb-induced Ewing cell death in the presence of human effector cells and serum complement. Published studies demonstrate a paucity of CD3+ lymphocytes in human Ewing sarcoma tumors, but an abundance of CD68+ tumor-associated macrophages (TAMs) and increased levels of IL-6 in patients with poor clinical outcomes.22-25 Apart from this, the understanding of the human Ewing sarcoma immune milieu remains limited. As described herein, a humanized Ewing sarcoma mouse model can be used to better understand the tumor immune milieu, mAb- induced cytotoxicity within the human immune context, and on-target off-tumor binding. CD99 expression is greatest on human leukocytes and pancreatic beta islet cells underscoring the importance of on-target off-tumor study as a means by which to anticipate toxicity.19 Without wishing to be bound by theory, results from these experiments will identify an anti- CD99 immunotherapeutic that is more broadly applicable to other CD99-positive tumors (such as additional sarcomas, leukemias, lymphomas, and prostate tumors). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00239] Experimental Data [00240] Phagemid panning utilizing a 27 billion member human single-chain variable fragment (scFv)-phage library (S. Mehta and W.A. Marasco) was performed for three rounds against soluble human CD99. The concentration of soluble CD99 was serially decreased with each round to a minimum of 1μg/mL. Rescued phage were analyzed for CD99 binding by ELISA with the detection of 75 binding clones. Phagemid sequencing of these clones revealed 10 unique scFv antibodies. Fluorescence activated cell sorting (FACs) was performed to analyze binding of these scFv’s to patient-derived Ewing sarcoma cell lines and the resultant six binding scFv’s were screened by ELISA against serial dilutions of soluble CD99 to identify the top three clones with reproducible, differential binding at concentrations of 0.5μg/mL or less (FIG.1). Sequences for the top three human anti-CD99 scFv heavy and light chains (termed clones 1,2, and 3) were cloned into an IgG1 vector for transfection of 293F cells. Resultant antibodies were purified by protein A and analyzed for in vitro binding by FACs to 5 patient-derived Ewing sarcoma cell lines. Serial dilutions of each antibody were applied to Ewing cells in a 96-well plate demonstrating dose-dependent binding and retained affinity at low concentrations (FIG.2). OctetRED (Biacore) was utilized to determine the equilibrium dissociation constant (KD) for each clone (~4nM). Cell proliferation and viability studies were performed utilizing CellTiter-Glo and MTT assays. Cell death was not observed in isolation (i.e in the absence of immune effector cells), nor did CD99 internalize following antibody engagement. To assess effector cell and complement mediated CD99-propagated cell death, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) experiments were performed.26,27 Reproducible Ewing cell death was demonstrated in both assays in a mAb concentration- dependent fashion across three patient-derived cell lines. The effect appeared greatest for p53 wild-type lines (CADO-ES1, TC32) (FIG.3). Clones 2 and 3 demonstrated binding by FACs at low mAb concentrations and more effective mAb induced cytotoxicity. Therefore, these clones will be utilized as representative clones for in vivo studies going forth. [00241] Experimental design [00242] Aim 1(a): To examine the Ewing sarcoma tumor immune environment in a humanized Ewing sarcoma xenograft model pre- and post-treatment with human anti-CD99 mAb. [00243] In vitro data demonstrates the importance of the immune system in propagating Ewing cell death following human anti-CD99 mAb engagement. Use of a humanized mouse DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 for in vivo experiments will allow study of the tumor immune environment, categorizing immune elements present before and after treatment with human anti-CD99 mAb. [00244] Methods: [00245] Humanized mouse models: Neonatal Nod SCID gamma (NSG) mice will undergo whole-body irradiation (100cGy) and subsequent intrahepatic injection of 3x104 CD34+ hematopoietic stem cells (HSCs). After 12 weeks, flow cytometric analyses of the blood will be performed to ensure engraftment defined as >20% peripheral human CD45+ cells. Previous studies have shown successful growth of patient-derived xenografts 2-12 weeks following HSC injection.28 For the purposes of this study, mice will be injected with Ewing sarcoma patient-derived cells (TC32) 8-10 weeks post-receipt of HSCs, utilizing previously vetted approaches for the development of subcutaneous and orthotopic tumors. More specifically, mice will be injected subcutaneously on the right flank with 1 million Ewing sarcoma cells re-suspended in 100uL of PBS. Tumor size will be measured serially with calipers until tumors reach 100mm3 with an anticipated time to tumor growth of 3-4 weeks. Ewing sarcoma orthotopic models will be pursued by intratibial injection of 50,000 Luc-mCh transduced cells (allowing imaging by bioluminescence, BLI) re-suspended in 50uL (50% matrigel, 50% PBS) with a 3-4 week anticipated time frame to tumor formation. [00246] Evaluation of the tumor immune microenvironment: A cohort of mice will be euthanized at 14-18 weeks (i.e.2-4 weeks following tumor formation), and the tumor immune microenvironment interrogated by immunohistochemistry (IHC), flow cytometry of single cell suspensions, and Nanostring Technology®.29 IHC and flow cytometric analyses will examine cell surface targets representative of a range of immune cells (FIG.4). Finally, to functionally characterize the immune milieu, RNA will be extracted from tumor tissue and a Cancer Immune Nanostring Technology® panel run to examine 760 gene transcripts relevant to tumor infiltrating immune cells and cytokines/chemokines. nSolver™ software will be used to identify transcripts with significant enrichment. A cohort of mice will be treated with 3mg/kg of antibody administered via tail vein twice weekly for 3-5 weeks (see Aim 1(b)). Mice will be euthanized at staggered time points post-treatment and tumors analyzed herein, allowing a comparison of the immune environment pre- and post-therapy. [00247] Anticipated results: [00248] Existing data is available regarding the timeline to xenograft growth for both subcutaneous and orthotopic tumors, however this timeline can vary in a humanized mouse. Timing of tumor cell inoculates and pre- and post-treatment assessments can require adjustment depending on the success of xenograft growth and the onset of graft-versus-host DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 disease (FIG.5). To address this uncertainty, a large cohort of mice will be injected with HSC’s from the outset, to allow staggered timing of tumor inoculate and tumor immune assessments but also to allow parallel work on Aims 1(b) and 1(c). Subcutaneous patient- derived xenografts grow successfully for a variety of tumor types in humanized mice. While orthotopic tumors would provide unique information regarding the tumor immune milieu, subcutaneous models can be prioritized if they prove the most reliable. [00249] Aim 1(b): To determine the effect of treatment with human anti-CD99 mAb on tumor cell growth. [00250] Ewing cell death in vitro following anti-CD99 mAb engagement relies upon the presence of immune effector cells or serum complement. Use of a humanized mouse model will allow a corresponding study of mAb-induced Ewing cell death in vivo in the context of the human immune environment. [00251] Methods: [00252] In vivo cytotoxicity: Humanized Ewing xenograft models with subcutaneous or orthotopic tumors (at 100mm3 or 4 weeks of growth, respectively) will be treated via tail vein with anti-CD99 mAb or an isotype control dosed at 3mg/kg twice weekly. Mice will be followed prospectively until tumor progression (>20% increase in size) or onset of graft- versus-host disease. Tumor burden will be serially assessed using calipers (subcutaneous tumors) and BLI (orthotopic tumors). A fraction of mice will undergo MRI to assess changes in tumor dimension, enhancement, or necrosis. Kaplan-Meier curves will be generated to determine whether treatment with anti-CD99 mAb impacts overall survival. [00253] Anticipated results: Without wishing to be bound by theory, anti-CD99 mAb alone or in combination with immune stimulating agents can serve as a therapeutic if experiments demonstrate slowed tumor growth or robust tumor cell death. Anti-CD99 mAb in combination with conventional chemotherapies can be a therapeutic agent if single-agent mAb treatment fail to impact tumor growth. Alternatively, human mAb drug or radionuclide delivery can ultimately require exploration. As noted, if orthotopic tumors are difficult to propagate in a humanized Ewing xenograft model, we will prioritize subcutaneous tumors for this arm of the study. [00254] Aim 1(c): To investigate on-target off-tumor binding of the human anti-CD99 mAb. [00255] The effects of off-tumor anti-CD99 mAb binding on leukocyte and pancreatic health remain unknown. As pancreatic beta cells express CD99 and are the source of insulin DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 production, damage to these cells can result in a clinical picture mimicking type I diabetes.19 Results to these experiments will further inform safety and efficacy in clinical development. [00256] Methods: Toxicity and preferential tumor binding will be assessed using a step- wise approach. [00257] Hematologic toxicity: Humanized mice will be created as per Aim 1(a) with HSC engraftment at 12 weeks verified by flow cytometry. Mice will then be injected with anti- CD99 mAb and serially bled for assessment of effect of anti-CD99 Ab binding on total white blood cell count. [00258] Pancreatic toxicity: Human fetal pancreatic cells at 18-24 weeks of gestation will be digested with collagenase and cultured as islet-like cell clusters (ICCs).30 The ICCs will be digested into single cells and transplanted under the kidney capsule of 6-week old non- humanized NSG mice. After three months, 1-2 representative mice will be euthanized with confirmation of subcapsular pancreatic tissue by IHC. Serum human peptide C levels will be measured as a proxy for insulin secretion. Mice will be injected with anti-CD99 mAb and serially bled to track serum peptide C and glucose levels. [00259] Preferential binding: Three cohorts of NSG mice will be prepared: 1) human blood-reconstituted, 2) human pancreatic tissue-engrafted, and 3) a combination of the two. Ewing sarcoma subcutaneous flank xenografts, which do not metastasize, will be established in each of the three cohorts. Once tumors reach ~100 mm3, mice will be injected with 64Cu- labeled anti-CD99 and imaged with PET; regions-of-interest will be drawn on marrow, pancreatic, and tumor tissue with standardized-uptake-values (SUVs) compared to assess competitive binding. Terminal radiotracer biodistribution studies will assess blood, pancreatic, tumor, and tissue radioactivity. Serum human peptide C levels will again be drawn in cohorts 2) and 3) to assess for pancreatic toxicity. Pending results from Specific Aim 1b, mice from each category can be treated with non-radionuclide bound antibody to assess for effect of competitive binding on tumor growth. Animal studies will follow Animal Care and Use Committee guidelines. [00260] Anticipated results: The translation of an anti-CD99 antibody to the clinic remains promising regardless of potential leukocyte cross-binding, as human subjects can be supported through hematologic toxicity much as they are with current treatments. While injury to the pancreatic beta islet cells, i.e. iatrogenic type I diabetes, is a serious potential toxicity, given the less than 20% survival of patients with metastatic, relapsed, or refractory Ewing sarcoma a risk of this nature can be warranted. Without wishing to be bound by theory, development of a bi-specific mAb (such as those targeting LINGO1, for example, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 which is reported unique to Ewing cells) can maximize on-tumor binding and preserve efficacy.31 [00261] References Cited In This Example 1. O'Neill AF, Dearling JL, Wang Y, et al. Targeted imaging of Ewing sarcoma in preclinical models using a 64Cu-labeled anti-CD99 antibody. Clinical cancer research: an official journal of the American Association for Cancer Research. Feb 12014;20(3):678-687. 2. Gellini M, Ascione A, Flego M, et al. Generation of human single-chain antibody to the CD99 cell surface determinant specifically recognizing Ewing's sarcoma tumor cells. Current pharmaceutical biotechnology.2013;14(4):449-463. 3. Guerzoni C, Fiori V, Terracciano M, et al. CD99 triggering in Ewing sarcoma delivers a lethal signal through p53 pathway reactivation and cooperates with doxorubicin. Clinical cancer research: an official journal of the American Association for Cancer Research. Jan 1 2015;21(1):146-156. 4. Cerisano V, Aalto Y, Perdichizzi S, et al. Molecular mechanisms of CD99-induced caspase-independent cell death and cell-cell adhesion in Ewing's sarcoma cells: actin and zyxin as key intracellular mediators. Oncogene. Jul 222004;23(33):5664-5674. 5. Gurney JG SA BM. Malignant Bone Tumors SEER Data Pediatrics.. National Cancer Institute 1975-1995. 6. Gurney JG YJ RS, Smith MA, Bunin GR. Soft Tissue Sarcomas SEER Data Pediatrics. National Cancer Institute 1975-1995. 7. Womer RB, West DC, Krailo MD, et al. Randomized controlled trial of interval- compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children's Oncology Group. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Nov 202012;30(33):4148-4154. 8. Grier HE, Krailo MD, Tarbell NJ, et al. Addition of ifosfamide and etoposide to standard chemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor of bone. The New England journal of medicine. Feb 202003;348(8):694-701. 9. Cotterill SJ, Ahrens S, Paulussen M, et al. Prognostic factors in Ewing's tumor of bone: analysis of 975 patients from the European Intergroup Cooperative Ewing's Sarcoma Study Group. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. Sep 2000;18(17):3108-3114. 10. Balamuth NJ, Womer RB. Ewing's sarcoma. The Lancet. Oncology. Feb 2010;11(2):184- 192. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 11. Donaldson SS. Ewing sarcoma: radiation dose and target volume. Pediatric blood & cancer. May 2004;42(5):471-476. 12. Liu AK, Stinauer M, Albano E, Greffe B, Tello T, Maloney K. Local control of metastatic sites with radiation therapy in metastatic Ewing sarcoma and rhabdomyosarcoma. Pediatric blood & cancer. Jul 152011;57(1):169-171. 13. Longhi A, Ferrari S, Tamburini A, et al. Late effects of chemotherapy and radiotherapy in osteosarcoma and Ewing sarcoma patients: the Italian Sarcoma Group Experience (1983- 2006). Cancer. Oct 152012;118(20):5050-5059. 14. Jain S, Kapoor G. Chemotherapy in Ewing's sarcoma. Indian journal of orthopaedics. Oct 2010;44(4):369-377. 15. Bernstein M, Kovar H, Paulussen M, et al. Ewing's sarcoma family of tumors: current management. The oncologist. May 2006;11(5):503-519. 16. Kovar H, Dworzak M, Strehl S, et al. Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor. Oncogene. Jul 1990;5(7):1067-1070. 17. Rocchi A, Manara MC, Sciandra M, et al. CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis. The Journal of clinical investigation. Mar 2010;120(3):668-680. 18. Weidner N, Tjoe J. Immunohistochemical profile of monoclonal antibody O13: antibody that recognizes glycoprotein p30/32MIC2 and is useful in diagnosing Ewing's sarcoma and peripheral neuroepithelioma. The American journal of surgical pathology. May 1994;18(5):486-494. 19. The Human Protein Atlas.:http://www.proteinatlas.org/. 20. Sankar S, Bell R, Stephens B, et al. Mechanism and relevance of EWS/FLI-mediated transcriptional repression in Ewing sarcoma. Oncogene. Oct 172013;32(42):5089-5100. 21. Franzetti GA, Laud-Duval K, Bellanger D, Stern MH, Sastre-Garau X, Delattre O. MiR- 30a-5p connects EWS-FLI1 and CD99, two major therapeutic targets in Ewing tumor. Oncogene. Aug 152013;32(33):3915-3921. 22. Hingorani P, Maas ML, Gustafson MP, et al. Increased CTLA-4(+) T cells and an increased ratio of monocytes with loss of class II (CD14(+) HLA-DR(lo/neg)) found in aggressive pediatric sarcoma patients. Journal for immunotherapy of cancer.2015;3:35. 23. Fujiwara T, Fukushi J, Yamamoto S, et al. Macrophage infiltration predicts a poor prognosis for human ewing sarcoma. The American journal of pathology. Sep 2011;179(3):1157-1170. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 24. Lissat A, Joerschke M, Shinde DA, et al. IL6 secreted by Ewing sarcoma tumor microenvironment confers anti-apoptotic and cell-disseminating paracrine responses in Ewing sarcoma cells. BMC cancer.2015;15:552. 25. Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nature reviews. Cancer. Jan 2004;4(1):71-78. 26. Chang DK, Sui J, Geng S, et al. Humanization of an anti-CCR4 antibody that kills cutaneous T-cell lymphoma cells and abrogates suppression by T-regulatory cells. Molecular cancer therapeutics. Nov 2012;11(11):2451-2461. 27. Chang DK, Kurella VB, Biswas S, et al. Humanized mouse G6 anti-idiotypic monoclonal antibody has therapeutic potential against IGHV1-69 germline gene-based B-CLL. mAbs. May-Jun 2016;8(4):787-798. 28. The Jackson Laboratory MS, Bar Harbor, ME, 04609. Non-HLA Matched Humanized NSG Mouse Model with Patient-Derived Xenograft. Patent: WO 2016/209865. December 2016. 29. Yu YR, O'Koren EG, Hotten DF, et al. A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues. PloS one.2016;11(3):e0150606. 30. Hayek A, Beattie GM. Experimental transplantation of human fetal and adult pancreatic islets. The Journal of clinical endocrinology and metabolism. Aug 1997;82(8):2471-2475. 31. Town J, Pais H, Harrison S, et al. Exploring the surfaceome of Ewing sarcoma identifies a new and unique therapeutic target. Proceedings of the National Academy of Sciences of the United States of America. Mar 292016;113(13):3603-3608. EXAMPLE 3 [00262] Methods [00263] In vitro cytotoxicity assays [00264] In vitro cell viability assays were conducted utilizing three different approaches: 1) serial cell counts, 2) a Cell Titer-Glo Luminescent Viability Assay, and 3) a Roche MTT cell proliferation assay. First, cells from 4 Ewing sarcoma cell lines (A673, TTC466, CADO-1, and TC32) were plated at 500,000 cells/plate in 15mLplates. Each plate was incubated with 0.5µg/mL of NOA 1,2, or 3 on day 1 and cell counts tallied at days 3, 5 and 7. For Cell Titer- Glo assays, 6 Ewing cells lines (A673, TC32, CADO-1, TC71, TTC466, and SKNEP-1, plus Kelly cells as a negative control) were plated on 384-well plates at 1,250 cells/well. Cells DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 were incubated with anti-CD99 or an IgG isotype control at concentrations ranging from 0.125µg/mL to 20µg/mL. Plate luminescence was quantified at days 0, 3 and 5. For MTT proliferation assays, 2 Ewing cell lines (A673 and TC32) were plated in a 96-well plate with 1µg/mL, 10µg/mL and 100µg/mL of anti-CD99 or an isotype control. Cell proliferation was assessed at 24 and 48 hours. [00265] Human effector cell and serum complement assays [00266] Antibody-dependent cell-mediated cytotoxicity (ADCC) assays were performed with the use of an ADCC Reporter Bioassay Core Kit (Promega #G7010, G7018). Ewing sarcoma cells were cultured overnight in a 96-well plate at 30,000 cells per well. Serial dilutions of anti-CD99 antibody clones or an isotype control were plated with cells the following morning at concentrations ranging from 0 µg/mL to 2 µg/mL. Effector cells were plated at an effector-to-target cell ratio of 2.5:1 and incubated for 6 hours at 37ºC with 5% CO2. A Bio-Glo Luciferase Assay was utilized to quantitate cell death. Plates were read on a POLARStar Omega plate reader and relative light units (RLU) analyzed for each condition after subtracting for background. [00267] Complement-dependent-cell (CDC) death assays were performed with pooled baby rabbit complement (Cedarlane). Ewing sarcoma cells were cultured overnight in a 96-well plate at 1x 106 cells per well. Serial dilutions of anti-CD99 antibody clones or an isotype control were plated with cells the following morning at concentrations ranging from 0.01 µg/mL to 10 µg/mL. Rabbit complement was added at a ratio of 1:8 or 1:16. CD20+ Raji cells were incubated with Rituximab as a control. Cells were incubated for 4 hours at 37ºC with 5% CO2. Lysis buffer was added to a control well to calculate 100% cytotoxicity. Cells were centrifuged at 4ºC for 4-5 minutes at 250g, supernatant was transferred to a new 96-well plate, assay reagent added and stop solution applied after 30 minutes. Plates were read on a Biorad microplate spectrophotometer at 490nm. % cytotoxicity was calculated using the following formula: ([Experimental – Effector Spontaneous – Target Spontaneous]/[Target Maximum – Target Spontaneous]) x 100. [00268] Antibody-dependent cellular phagocytosis (ADCP) assays were performed by first isolating human monocytes from human peripheral blood mononuclear cells (PBMCs) by plating PBMCs in MDM medium (DMEM with 10% heat-inactivated human serum, 1% penicillin/streptomycin, 1% L-glutamine, and 50ng/mL GM-CSF) in a Petri dish for 3 days at 37ºC with 5% CO2. At the end of three days, the supernatant was removed and MDM media replenished to further culture the adherent macrophage precursors. On day 7, macrophages were incubated with 0.5mM EDTA in PBS, lifted, and added at a ratio of 1:4 (target: effector DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 cell) to Ewing sarcoma cells stained with PKH26 fluorescent membrane dye (Sigma Aldrich). Isotype or anti-CD99 antibody was added to the wells at a concentration of 2µg/mL or 5µg/mL and incubated at 37ºC for 4 hours. After co-culture, cells were imaged using a Celigo plate reader and then lifted and transferred to a 96-well V-bottom plate for FACs. Engulfed target cells were defined as PKH26+/CD14+. [00269] Internalization [00270] 400,000 Ewing sarcoma cells were stained with DiD Lypophilic tracer (ThermoFisher Scientific), plated, and cultured overnight at 37ºC with 5% CO2 in phenol- free RPMI on uncoated MatTek coverslip-bottom 35mm dishes. The following day, cells were stained with biotin-avidin FITC-conjugated NOA2 at 2µg/mL and incubated for 2 hours, 1 hour and 0.5 hours at 37ºC with 5% CO2. Coverslips were washed with PBS three times and cells stained with Hoescht nuclear dye (ThermoFisher Scientific) at 1µg/mL 15 minutes prior to confocal imaging. Plates were imaged on an Andor Revolution Spinning Disc Confocal Microscope and images analyzed using Fiji ImageJ software. EXAMPLE 4 [00271] Results [00272] Isolation of CD99-specific scFv’s from phage display library [00273] Three non-immune human scFv-phage display libraries containing 12 (Mehta I) and 15 (Mehta II) billion members were combined and incubated with immunotube-bound CD99-Fc to isolate anti-CD99 specific scFv’s. Bound scFv’s were eluted from each panning round and screened by ELISA to confirm binding specificity. ELISA was first performed against a non-specific isotype IgG to subtract out Fc-region binders, and subsequently against soluble CD99-Fc. Twenty one of 144 clones from the first round of panning against 10µg/mL CD99-Fc and 29 of 192 clones from the second round of panning against 1µg/mL CD99-Fc specifically bound to CD99 as detected by ELISA. Of these 50 clones, DNA sequence analysis revealed that 10 were unique. Six of these phagemid scFv’s bound to A673 Ewing sarcoma cells by FACs. Binding affinity for these six scFv’s was assessed by ELISA with titration of soluble CD99-Fc concentrations from 0.03125 - 2 µg/mL. The top three clones, NOA1, 2, and 3 demonstrated reproducible binding at concentrations of 0.125µg/mL or less. These scFv’s were carried forth for further studies. The NOA1 heavy chain was of IGHV5-51*03, IGHD2-15*01, IGHJ3*02 germline origin and harbored 8 single-nucleotide mutations (SNMs). The NOA2 heavy chain was of IGHV5-51*03, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 IGHD1-26*01, IGHJ3*02 germline origin and harbored 8 SNMs and the NOA3 heavy chain was of IGHV5-51*01, IGHD3-16*01, IGHJ3*02 germline origin and harbored 7 SNMs. The NOA1 light chain was of IGLV3-1*01, IGLJ1*01 germline origin harboring 9 SNMs, the NOA2 light chain was of IGLV5-37*01, IGLJ3*02 germline harboring 7 SNMs and the NOA3 light chain was of IGLV3-1*01, IGLJ3*02 germline harboring 6 SNMs (FIG.14). [00274] Binding of anti-CD99 antibodies to Ewing sarcoma cells by flow cytometry [00275] NOA1, 2 and 3 were subcloned, expressed in 293F cells, and purified as IgG1 antibodies. The binding activity of these antibodies was further examined by flow cytometry to determine affinity for CD99+ patient-derived Ewing sarcoma cell lines. NOA1 demonstrated limited binding at concentrations as high as 10 µg/mL whereas NOA2 (light green) and 3 (dark green) demonstrated reproducible binding at concentrations of 2 µg/mL (FIG.6A). None of the clones bound to CD99-negative Kelly neuroblastoma cells (FIG. 6B). The relative affinity of NOA2 and 3 was determined by flow cytometric saturation binding studies against three independent CD99+ Ewing sarcoma cell lines. (A673 depicted in FIG.6C). Estimated EC50 was determined for each cell line by plotting the geometric mean fluorescence against concentration resulting in an EC50 of 0.125 µg/mL for NOA2 and 0.1 µg/mL for NOA3. [00276] Kinetic analysis of anti-CD99 antibody binding by OctetRED Analysis [00277] OctetRED analysis was pursued to determine the equilibrium dissociation constants (KD) of NOA2 and 3. Utilizing the OctetRED ForteBio Analysis Software Version 10.0, the KD for NOA2 was calculated at 3.35 x 10-9 M +/- 4.60 x 10-11 M from analysis of F(ab’)2 concentrations between 10 and 15nm with a Kon of 4.15 x 105 +/- 4.04 x 103 (1/Ms) and a Koff of 1.39 x 10-3 +/- 1.35 x 10-5 (1/s). The KD for NOA3 was calculated at 1.86 x 10-9 M +/- 1.65 x 10-10 M from analysis of F(ab’)2 concentrations ranging between 10 and 25nm with a Kon of 3.80 x 105 +/- 5.13 x 103 (1/Ms) and a Koff of 7.06 x 10-4 +/- 6.18 x 10-5 (1/s). (FIG.7). [00278] Anti-CD99 antibodies mediate killing of CD99+ Ewing sarcoma cells by ADCC and CDC [00279] In vitro viability and proliferation assays did not demonstrate antibody-mediated Ewing cell death in the absence of immune effector cells indicating that the immune system plays a key role in NOA antibody-mediated cytotoxicity. ADCC and CDC performed for three antibody clones demonstrated an increase in Ewing cell death as a function of antibody concentration. There was no Ewing cell death in lines incubated with an isotype control. As shown in FIG.8A-C NOA2 and 3 outperformed NOA1. Ewing cell death, as assessed by net DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 luminescence was more pronounced for the p53 wild- type cell lines CADO-ES1 and TC32. FIG.8D-F demonstrates cytotoxicity by CDC with three anti-CD99 antibody clones; NOA2 outperformed NOA1 and 3 at concentrations greater than 0.1 µg/mL. There was no CDC- mediated killing with incubation of Ewing cells with an isotype control. [00280] NOA2-mediated CD99 internalization [00281] Confocal microscopy was utilized to evaluate CD99 internalization (FIG.9). Live TC32 Ewing cells were stained with a lipophilic membrane dye, plated, and subsequently incubated with FITC-conjugated NOA2 mAb for 0.5, 1 and 2 hours. Nuclear Hoescht stain was added just prior to live cell confocal imaging. Independent of duration of incubation, FITC-bound CD99 was visualized in the cytoplasm, at multiple imaging planes, co-localizing with Did dye indicating NOA2-propagated endocytosis of membrane-bound CD99. Time- lapse microscopy demonstrated intracellular migration of these dual-fluorescent intracytoplasmic bodies. [00282] In vivo Ewing tumor growth arrest following treatment with NOA2 [00283] NSG mice with Ewing sarcoma micro-metastases, established via tail vein injection, were injected with human peripheral blood mononuclear cells (PBMCs) and treated with NOA2 (n=3) or an isotype control (n=2) twice weekly for two weeks. FIG.10 demonstrates tumor growth arrest, as extrapolated by mean luminescence, in mice treated with NOA2 as compared with an isotype control. NOA2 utilized for these studies on the basis of FACs, OctetRED, and ADCC/CDC data indicating superior binding and Ewing tumor cell kill. Following therapy, mice were euthanized, livers dissected, embedded in paraffin, and sectioned. Hematoxylin-eosin staining demonstrated pockets of central necrosis positive for apoptotic bodies (TUNEL) in tumors of mice treated with NOA2. This was not observed in mice treated with isotype control. Tumor specimens stained only weakly for human CD45+ cells. However, in contrast to mice treated with an isotype control, tumors from mice treated with NOA2 demonstrated a pronounced mouse myeloid cell infiltrate (mouse CD14+). Tumors from mice treated with NOA2 demonstrated upregulation of IGF- 1R and Ras implying activation of the IGF-1R/Ras/Rac1 pathway. [00284] Anti-CD99 antibodies mediate killing of CD99+ Ewing sarcoma cells by ADCP [00285] On the basis of in vivo immunohistochemistry results demonstrating a prominent myeloid infiltrate, ADCP studies were performed. Marophages isolated from human PBMCs co-incubated with Ewing sarcoma cells and anti-CD99 antibody demonstrate changes in morphology indicative of activation and tumor cell engulfment for p53 wild-type cell lines CADO-ES1 and TC32 (FIG.11). FACs demonstrates an increase in PKH26+/CD14+ cells, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 representing Ewing cells engulfed by macrophages, following exposure to anti-CD99; this does not appear antibody dose-dependent. While there are morphologic changes in macrophage appearance and engulfment of Ewing sarcoma cells in the presence of isotype IgG, these findings are far more pronounced for CADO-ES1 and TC32 lines in the presence of anti-CD99. [00286] Macrophage activation following co-incubation with Ewing sarcoma cells and anti-CD99, anti- PILR^ antibodies [00287] ELISA results demonstrate a dose-dependent binding of PILR^ to soluble CD99 (FIG.12A) and FACs results reflect competitive binding between PILR^ and anti-CD99, indicating a shared epitope on CD99 (FIG.12B). Our next series of experiments were aimed at interrupting the PILR^:CD99 macrophage:Ewing sarcoma binding axis to assess affects of this intervention on macrophage activiation, using TNF-^ secretion as a proxy. FIG.12C illustrates the PILR^:CD99 axis and the postulated role of anti-CD99 and anti- PILR^ in interrupting macrophage:Ewing sarcoma cell interaction. When macrophages were incubated with anti-CD99 or anti- PILR^ antibodies alone, there was little difference in TNF- ^ secretion at each timepoint of supernatant collection; however when macrophages were incubated with both antibodies, there was a substantial decrease in TNF-^ secretion and by proxy, macrophage activity (left graph, FIG.12D). When macrophages were pre-incubated with TC32 Ewing sarcoma cells and anti-CD99 or anti- PILR^ antibody, a similar pattern was noted for TNF-^ as that seen with macrophages alone. However, when macrophages plus tumor cells were incubated with the combination of antibodies, a recovery of macrophage function/activity was be appreciated with elevation in TNF-^ levels at timepoints (right graph, FIG.12D). [00288] In vitro Migration Studies [00289] In vitro migration studies (FIG.13) indicating that even in the presence of antibody-mediated CD99 blockade, leukocytes traffic through a single-cell layer of endothelium albeit to a lesser extent than with following treatment with isotype IgG. [00290] References Cited In This Example 1. Gurney, J. G., Severson, R. K., Davis, S. & Robison, L. L. Incidence of cancer in children in the United States. Sex-, race-, and 1-year age-specific rates by histologic type. Cancer 75, 2186-2195 (1995). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 2. Womer, R. B. et al. Randomized controlled trial of interval-compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children's Oncology Group. J Clin Oncol 30, 4148-4154, doi:10.1200/JCO.2011.41.5703 (2012). 3. Grier, H. E. et al. Addition of ifosfamide and etoposide to standard chemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor of bone. N Engl J Med 348, 694-701, doi:10.1056/NEJMoa020890 (2003). 4. Cotterill, S. J. et al. Prognostic factors in Ewing's tumor of bone: analysis of 975 patients from the European Intergroup Cooperative Ewing's Sarcoma Study Group. J Clin Oncol 18, 3108-3114, doi:10.1200/JCO.2000.18.17.3108 (2000). 5. Longhi, A. et al. Late effects of chemotherapy and radiotherapy in osteosarcoma and Ewing sarcoma patients: the Italian Sarcoma Group Experience (1983-2006). Cancer 118, 5050-5059, doi:10.1002/cncr.27493 (2012). 6. Balamuth, N. J. & Womer, R. B. Ewing's sarcoma. Lancet Oncol 11, 184-192, doi:10.1016/S1470-2045(09)70286-4 (2010). 7. Donaldson, S. S. Ewing sarcoma: radiation dose and target volume. Pediatr Blood Cancer 42, 471-476, doi:10.1002/pbc.10472 (2004). 8. Jain, S. & Kapoor, G. Chemotherapy in Ewing's sarcoma. Indian J Orthop 44, 369-377, doi:10.4103/0019-5413.69305 (2010). 9. Rocchi, A. et al. CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis. J Clin Invest 120, 668-680, doi:10.1172/JCI36667 (2010). 10. Bernstein, M. et al. Ewing's sarcoma family of tumors: current management. Oncologist 11, 503-519, doi:10.1634/theoncologist.11-5-503 (2006). 11. Cerisano, V. et al. Molecular mechanisms of CD99-induced caspase-independent cell death and cell-cell adhesion in Ewing's sarcoma cells: actin and zyxin as key intracellular mediators. Oncogene 23, 5664-5674, doi:10.1038/sj.onc.1207741 (2004). 12. Kovar, H. et al. Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor. Oncogene 5, 1067-1070 (1990). 13. Weidner, N. & Tjoe, J. Immunohistochemical profile of monoclonal antibody O13: antibody that recognizes glycoprotein p30/32MIC2 and is useful in diagnosing Ewing's sarcoma and peripheral neuroepithelioma. Am J Surg Pathol 18, 486-494 (1994). 14. Franzetti, G. A. et al. MiR-30a-5p connects EWS-FLI1 and CD99, two major therapeutic targets in Ewing tumor. Oncogene 32, 3915-3921, doi:10.1038/onc.2012.403 (2013). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 15. Manara, M. C. et al. CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling. Oncotarget 7, 79925-79942, doi:10.18632/oncotarget.13160 (2016). 16. Parija, T. et al. Type 1 (11;22)(q24:q12) translocation is common in Ewing's sarcoma/peripheral neuroectodermal tumour in south Indian patients. J Biosci 30, 371-376 (2005). 17. Vural, C., Uluoglu, O., Akyurek, N., Oguz, A. & Karadeniz, C. The evaluation of CD99 immunoreactivity and EWS/FLI1 translocation by fluorescence in situ hybridization in central PNETs and Ewing's sarcoma family of tumors. Pathol Oncol Res 17, 619-625, doi:10.1007/s12253-010-9358-3 (2011). 18. Ventura, S. et al. CD99 regulates neural differentiation of Ewing sarcoma cells through miR-34a-Notch-mediated control of NF-kappaB signaling. Oncogene 35, 3944-3954, doi:10.1038/onc.2015.463 (2016). 19. Gellini, M. et al. Generation of human single-chain antibody to the CD99 cell surface determinant specifically recognizing Ewing's sarcoma tumor cells. Curr Pharm Biotechnol 14, 449-463 (2013). 20. Crompton, B. D. et al. The genomic landscape of pediatric Ewing sarcoma. Cancer Discov 4, 1326-1341, doi:10.1158/2159-8290.CD-13-1037 (2014). 21. Atlas, T. H. P. (https://www.proteinatlas.org). 22. Martens, G. A., De Punt, V. & Stange, G. CD99 as surface anchor for human islet endocrine cell purification. J Tissue Eng Regen Med, doi:10.1002/term.2329 (2016). 23. O'Neill, A. F. et al. Targeted imaging of Ewing sarcoma in preclinical models using a 64Cu-labeled anti-CD99 antibody. Clin Cancer Res 20, 678-687, doi:10.1158/1078- 0432.CCR-13-1660 (2014). 24. Lou, O., Alcaide, P., Luscinskas, F. W. & Muller, W. A. CD99 is a key mediator of the transendothelial migration of neutrophils. J Immunol 178, 1136-1143 (2007). 25. Hingorani, P. et al. Increased CTLA-4(+) T cells and an increased ratio of monocytes with loss of class II (CD14(+) HLA-DR(lo/neg)) found in aggressive pediatric sarcoma patients. J Immunother Cancer 3, 35, doi:10.1186/s40425-015-0082-0 (2015). 26. Fujiwara, T. et al. Macrophage infiltration predicts a poor prognosis for human ewing sarcoma. Am J Pathol 179, 1157-1170, doi:10.1016/j.ajpath.2011.05.034 (2011). 27. Lissat, A. et al. IL6 secreted by Ewing sarcoma tumor microenvironment confers anti- apoptotic and cell-disseminating paracrine responses in Ewing sarcoma cells. BMC Cancer 15, 552, doi:10.1186/s12885-015-1564-7 (2015). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 EXAMPLE 5 [00291] As described herein, an anti-CD99 antibody, NOA2, binds Ewing sarcoma cells with high affinity. NOA2 can induce Ewing cell death through engagement of macrophages and antibody-mediated cellular phagocytosis in vitro. Tumors from humanized xenograft mice treated with NOA2 arrest growth and contain morphologically activated, intratumoral infiltrates of both mouse and human macrophages. Furthermore, inhibition of the binding of Ewing CD99 to macrophage PILR^, an inhibitory receptor and CD99 ligand, by dual anti- CD99 and anti-PILR^ blockade fosters macrophage reactivation. Without wishing to be bound by theory, binding of CD99 by NOA2 promotes both immune-mediated killing of Ewing cells and immunomodulation of the tumor microenvironment, more specifically, the myeloid cell compartment. [00292] RESULTS [00293] Binding of anti-CD99 antibodies to Ewing sarcoma cells by flow cytometry. Three anti-CD99 antibodies (NOA1, 2, and 3) were isolated by phage display and characterized (FIG.14 and Table 12). Table 12. Germline Segment Usage and Single Nucleotide Mutations (SNM) for three binding anti-CD99 scFv’s (NOA1, 2, and 3). *SNM tabulation excludes the 5’ end mutations as these are attributed to substitutions introduced during priming. mAb VH DH JH #SNM* [00294] FACs binding of IgG1 isoforms to CD99-positive patient-derived Ewing sarcoma cell lines was performed (FIG.15). NOA1 (blue) demonstrated limited binding at concentrations as high as 10 µg/mL whereas NOA2 (red) and 3 (green) demonstrated strong DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 binding at concentrations of 2 µg/mL (FIG.15A (upper)). None of the clones bound to CD99-negative Kelly neuroblastoma cells (FIG.15A (lower)). NOA2 was carried forth for further studies given superior binding across Ewing cell lines (results not shown). The relative affinity of NOA2 was determined by flow cytometric saturation binding studies against five independent CD99-positive Ewing sarcoma cell lines (FIG.15B). Estimated EC50 was determined by plotting the geometric mean fluorescence against concentration (FIG.15C). [00295] Anti-CD99 antibodies mediate killing of CD99+ Ewing sarcoma cells by ADCC and ADCP. In vitro viability and proliferation assays did not result in direct antibody-mediated Ewing cell death in the absence of immune effector cells. To determine if immune effector cells can play a key role in NOA2 antibody-mediated cytotoxicity, ADCC and ADCP were performed. NOA2 triggered Ewing cell death as a function of antibody concentration in ADCC assays, however the magnitude of cell killing was cell line specific. As shown in FIG.16A-C, Ewing cell death as assessed by net luminescence was more pronounced for the p53 wildtype cell lines CADO-ES1 and TC32. Ewing cells retained viability when incubated with an isotype control. [00296] For in vitro ADCP studies, macrophages isolated from human PBMCs and co- incubated with Ewing sarcoma cells and NOA2 showed aggregation and engulfment of tumor cells (FIG.16D, arrows). Similar to ADCC, these findings were limited to p53 wildtype cell lines CADO-ES1 and TC32. FACs demonstrated an increase in PKH26+/CD14+ cells, representing Ewing cells engulfed by macrophages following NOA2 incubation; the two NOA2 concentrations tested for ADCP (2 & 5 µg/ml) did not show an antibody dose- dependent effect (FIG.16E). ADCP was most prominent following incubation with 2 µg/mL indicating that lower concentrations of NOA2 can be more effective for this immune effector function. There was comparatively minimal aggregation and engulfment of Ewing sarcoma cells with incubation of isotype IgG. [00297] In vivo Ewing tumor growth arrest following treatment with NOA2 and mechanism(s) of action. Whether anti-CD99 antibody treatment of mice harboring micro- metastatic Ewing sarcoma tumors can inhibit tumor growth was next determined. A human peripheral blood leukocyte (PBL)-NSG humanized mouse model with Ewing sarcoma micro- metastases was established and mice were treated with NOA2 (n=3) or an isotype control (n=2), initiated 24 hours after PBMC injection, for a total of 4 treatments (arrows). FIG.17A demonstrates tumor growth arrest at 14 days post-treatment, as extrapolated by mean luminescence, in mice treated with NOA2 as compared with an isotype control. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00298] To investigate NOA2’s mode of action, mice were euthanized following therapy and livers dissected, embedded in paraffin, and sectioned (FIG.17B). The representative images demonstrate a more prominent human CD45, mouse CD14, and human CD33 and CD16 infiltrate. There was a statistically significant difference in human CD45+, mouse CD14+, and human CD33+ infiltrating cells (p<0.05) in mice treated with IgG vs. NOA2 when averaged over three 10x high-powered fields. The differences for human CD16+ cells (p<0.16) did not reach statistical significance, due to small tumors and low overall cell counts (FIG.17C). There was a noticeable difference in the appearance of mouse myeloid cells staining for CD14; these cells were more plump and aggregated than those visualized in IgG- treated tumors indicating macrophage activation, similar to that seen in in vitro ADCP studies. Anti-CD99 treated tumors also demonstrated pockets of necrosis indicative of apoptosis by TUNEL staining, and marked upregulation of IGF-1, IGF-1R and Ras implicating activation of the IGF-1R/Ras/Rac-1 pathway (FIG.17B). Finally, on the basis of in vivo immunohistochemistry results demonstrating an upregulation of IGF-1, we sought to determine whether IGF-1 can recruit monocytes to the tumors. MCP-1 was utilized as a positive control in a transwell assay (FIG.18A, left panel). As shown in FIG.18B, right panel, a statistically significant increase in monocyte chemotaxis was observed at IGF-1 doses greater than 10 ng/mL [00299] Human CD33+ tumoral infiltrate as a function of NOA2 therapy. The observation that treatment with NOA2 led to an accumulation of hCD45+ and hCD33+ cells in Ewing tumors and an apparent activation of mouse CD14+ myeloid cells led us to perform a second treatment study in CD34+ HSC reconstituted NSG-SGM3 mice which show enhanced engraftment of human myeloid cells.P25,P26 Following treatment with IgG vs. NOA2, human CD45+ cells were isolated from subcutaneous tumors and analyzed by FACs. Cells were gated for human CD45+ expression; the percentage of CD45+ cells expressing human CD33+ at baseline compared to three timepoints throughout therapy was reported graphically. As shown in FIG.19 there is a robust CD33+ myeloid cell infiltrate in NOA2- treated mice with ~55% of hCD45+ cells being derived from the myeloid lineage (p<0.01). In summary, these collective results demonstrate that NOA2 treatment of Ewing tumors leads to the recruitment of blood monocytes/myeloid cells which can participate in tumor cell killing through ADCC and ADCP. [00300] Macrophage activation following co-incubation of Ewing sarcoma cells with anti-CD99 and anti- PILR^ antibodies. PILR^ is a CD99 ligand, and inhibitory receptor, DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 that is preferentially expressed on human myeloid cells but also on natural killer cells, dendritic cells, and other granulocytes. PILRα has been implicated in and can be restrictive of immune cell recruitment and activation.P27-P30 Data in FIG.20A confirms dose-dependent binding of our synthesized human PILR^ to soluble human CD99 by ELISA. PILR^ and NOA2 was next shown to bind Ewing CD99 competitively by FACs indicating a shared CD99 binding epitope (FIG.20B). FACs results demonstrate two distinct cell populations of PILR^ binding, the second of which is more impacted by anti-CD99 dose-escalation. The next series of experiments were aimed at using NOA2 and anti-PILR^ antibody to interrupt the PILR^:CD99, macrophage:Ewing sarcoma, axis to assess effects on macrophage activity, using TNF-^ secretion as a proxy. As shown in FIG.20C, TNF-^ secretion wanes naturally over the course of 48 hours from polarized (M1) macrophages incubated in vitro. Treatment of these macrophages with anti-CD99 or anti-PILR^ antibody alone did not lead to a change in TNF-^ secretion at each timepoint compared to untreated M1 macrophages. When M1 macrophages were incubated with Kelly CD99-negative neuroblastoma cells and treated with both anti-CD99 or anti-PILR^ antibodies, there was a decline in TNF-^ secretion at time points comparable to findings seen when M1 macrophages were incubated with both antibodies in the absence of Kelly cells (FIG.20C, left panel). When polarized macrophages were cultured with TC32 Ewing sarcoma cells, and exposed to both anti-CD99 and anti- PILR^ antibodies, a statistically significant rebound in the concentration of TNF-^ was observed across time points (FIG.20C, right panel) indicating that dual blockade of the inhibitory Ewing CD99:macrophage PILR^ axis, i.e. a checkpoint pathway unique to this macrophage: tumor interaction, is associated with recovery of macrophage TNF^ secretion. Further studies will be conducted to analyze the anti-tumor outcome of this combination immunotherapy in vivo. [00301] Isolation of CD99-specific scFv’s from a phage display library and their genetic analysis. Two non-immune human scFv-phage display libraries containing 12 (Mehta I) and 15 (Mehta II) billion members were combined and incubated with immunotube-bound CD99-Fc. Bound scFvs were eluted from each panning round and screened by ELISA to confirm binding specificity. ELISA was first performed against an isotype-matched human monoclonal IgG1 to subtract out Fc-region binders, and subsequently against soluble CD99-Fc. Of the 50 resultant clones, DNA sequence analysis revealed that 10 were unique. Six of these phagemid scFvs bound to A673 Ewing sarcoma cells by FACs. Binding affinity for these six scFv’s was assessed by ELISA with titration of soluble CD99- DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 Fc concentrations. The top three clones, NOA1, 2, and 3 demonstrated saturable binding at concentrations of 0.125µg/mL or less. These scFvs were carried forth for further studies. [00302] The germline Ig gene segment usage and somatic hypermutation (SHM) are shown in FIG.14 and Table XX. The NOA2 heavy chain is most closely aligned with the *03 allele of the IGHV5-51 germline gene, the *01 allele of the IGHD1-26 germline gene, and the *02 allele of the IGHJ3 germline segment. The heavy chain harbors 7 single-nucleotide mutations (SNMs) including in CDR1, however CDR2 is in the germline configuration. The light chain utilizes IGLV5-37*01 and IGLJ3*02 germline genes and has 6 SNM. [00303] Kinetic analysis of anti-CD99 antibody binding by OctetRED Analysis. OctetRED analysis was performed to determine the equilibrium dissociation constants (KD) of NOA2 and 3 when bound to soluble CD99. Utilizing the OctetRED ForteBio Analysis Software Version 10.0, the KD for NOA2 was calculated at 3.35 x 10-9 M +/- 4.60 x 10-11 M with Kon of 4.15 x 105 +/- 4.04 x 103 (1/Ms) and Koff of 1.39 x 10-3 +/- 1.35 x 10-5 (1/s). The KD for NOA3 was calculated at 1.86 x 10-9 M +/- 1.65 x 10-10 M with Kon of 3.80 x 105 +/- 5.13 x 103 (1/Ms) and Koff of 7.06 x 10-4 +/- 6.18 x 10-5 (1/s). (FIG.23). In summary, both antibodies bind strongly to CD99, and the circa 2-fold higher affinity of NOA3 over NOA2 is almost entirely due to the faster Koff for NOA2. [00304] DISCUSSION [00305] NOA2, a human monoclonal antibody that targets CD99 can recruit and reactivate components of the innate immune system to direct antibody-mediated Ewing sarcoma death both in vitro and vivo. NOA2 can trigger antibody-mediated tumor death through ADCC (Fc^R-mediated killing by natural killer cells) and ADCP (phagocytosis by macrophages). In vivo treatment of Ewing tumor-bearing mice with NOA2 prompts an enhanced infiltrate of morphologically activated mouse CD14+ cells, along with an increase in intratumoral human CD33+ cells and tumor growth arrest and apoptosis. NOA2 is also associated with upregulation of IGF-1, potentially contributing to monocyte/macrophage chemotaxis and recruitment. Combination NOA2 and PILRα blockade in vitro reverses the inhibitory CD99:PILRα pathway linking Ewing sarcoma cells and macrophages leading to restoration of TNF-α secretion. In light of published data showing M2 tumor-protective macrophages to be the predominant infiltrating immune subtype in human Ewing tumors, an antibody with the capacity to recruit and engage macrophages, as well as reactivate tumor-resident macrophages, can meaningfully altering outcomes.P19,P20 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00306] Several immune-mediated mechanisms of action were investigated to further understand the tumoricidal activities of NOA2. The differential ability of NOA2 to induce ADCC and ADCP in p53 wild-type vs. mutant cell lines was observed. Scotlandi and colleagues have demonstrated that anti-CD99 induced apoptosis is wild-type p53 dependent.P11 Without wishing to be bound by theory, wild type p53 is also relevant for cytotoxicity downstream of Fc^R-engagement. The differences in ADCP killing were reconciled by examining the overexpression of CD47, recognizing that the CD47/SIRP^ axis controls myeloid cell activation.P31 At baseline, human Ewing-derived cell lines express CD47; a more dramatic upregulation of CD47 on the A673 mutant p53 was noted following treatment with NOA2 potentially inhibiting the success of ADCP (FIG.21). p53 mutation status fortunately is of limited translational concern, given that the vast majority of Ewing tumors in humans are p53 wildtype at diagnosis.P32 [00307] There is a documented relationship between the Ewing EWS:FLI1 fusion protein and its role in enhancing transcription of insulin growth factor-1 (IGF-1).P33 Without wishing to be bound by theory, binding of NOA2 can trigger, through agonist activity, downstream signaling leading to upregulation of EWS:FLI1 expression which in turn increases IGF-1 transcription (FIG.22). IGF-1 serves as a monocyte chemoattractant which explains the more prominent myeloid infiltrate visualized in NOA2- vs. IgG-treated tumors from the in vivo studies. Mouse CD14+ cells from our initial in vivo experiment were morphologically activated, similar to those seen in our ADCP studies. On the basis of these findings, NOA2 is responsible for recruiting activated, intratumoral macrophages that are contributing to tumor growth arrest via ADCP. Cells staining for human CD16 were also present in higher numbers in NOA2-treated tumors. CD16 is expressed both on a subset of monocytes and NK cells; both CD16-expressing monocytes and NK cells are capable of ADCC, an additional contributor to in vivo tumor apoptosis.P34 The study of tumor-infiltrating lymphocytes has received considerable attention, given the striking successes in use of PD-1 inhibitors for adult malignancies. However, these agents are less likely to be of benefit in Ewing sarcoma given that Ewing tumors are mutationally silent and harbor a paucity of T-cells.P18-P20,P32 Pivoting focus towards manipulation of the myeloid compartment is of therapeutic relevance. [00308] While NOA2 can serve to recruit activated macrophages to the intratumoral microenvironment, tumor-protective macrophages are present.P19,P20 The exploration of the PILR^:CD99 axis addresses whether dormant macrophages can be re-activated through disruption of PILR^:CD99 binding. Human PILR^ binds to human CD99 and that NOA2 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 blocks this interaction.P35 Macrophages are known to express both surface PILR^ and CD99; co-incubation with saturating concentrations of anti-PILR^ and CD99 antibodies in this setting leads to binding of both of these receptors on the macrophage cell surface and CD99 on the Ewing cell surface (FIG.20D). In studies utilizing a CD99 negative Kelly neuroblastoma cell line, repression of TNF-^ secretion is observed at timepoints of antibody incubation. Introduction of Ewing CD99+ cells to polarized macrophages introduces a new Ewing:macrophage, CD99: PILR^, binding axis predicted to contribute to macrophage suppression. Interruption of this axis by dual antibody blockade reverses this inhibitory pathway allowing macrophages to again secrete TNF-^. These data show that a second target of PILR^, other than CD99, exists on Ewing cells and that anti-PILR^ blockade contributes to restoration of TNF-^ secretion. The dual FACs peak noted in FIG.20B points to this accessory pathway. [00309] In summary, this is the first study to demonstrate the role of human immune cells in antibody-mediated Ewing cell death and to identify a new immune axis capable of reactivating tumor-associated macrophages. For the population of Ewing sarcoma patients with metastatic, relapsed, or refractory disease, there is a grave need for new therapeutics. For patients with single-site disease, cure still comes at the expense of significant long-term toxicities. In the era of immunotherapy, an antibody that targets a heavily expressed membrane-associated tumor antigen and recruits and reactivates immune cells in the tumor microenvironment can provide a less toxic, more efficacious therapy for Ewing sarcoma patients. [00310] REFERENCES (P) [00311] P1 Gurney, J. G., Severson, R. K., Davis, S. & Robison, L. L. Incidence of cancer in children in the United States. Sex-, race-, and 1-year age-specific rates by histologic type. Cancer 75, 2186-2195 (1995). [00312] P2 Womer, R. B. et al. Randomized controlled trial of interval- compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children's Oncology Group. J Clin Oncol 30, 4148-4154, doi:10.1200/JCO.2011.41.5703 (2012). [00313] P3 Grier, H. E. et al. Addition of ifosfamide and etoposide to standard chemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor of bone. N Engl J Med 348, 694-701, doi:10.1056/NEJMoa020890 (2003). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00314] P4 Cotterill, S. J. et al. Prognostic factors in Ewing's tumor of bone: analysis of 975 patients from the European Intergroup Cooperative Ewing's Sarcoma Study Group. J Clin Oncol 18, 3108-3114, doi:10.1200/JCO.2000.18.17.3108 (2000). [00315] P5 Longhi, A. et al. Late effects of chemotherapy and radiotherapy in osteosarcoma and Ewing sarcoma patients: the Italian Sarcoma Group Experience (1983- 2006). Cancer 118, 5050-5059, doi:10.1002/cncr.27493 (2012). [00316] P6 Balamuth, N. J. & Womer, R. B. Ewing's sarcoma. Lancet Oncol 11, 184-192, doi:10.1016/S1470-2045(09)70286-4 (2010). [00317] P7 Donaldson, S. S. Ewing sarcoma: radiation dose and target volume. Pediatr Blood Cancer 42, 471-476, doi:10.1002/pbc.10472 (2004). [00318] P8 Jain, S. & Kapoor, G. Chemotherapy in Ewing's sarcoma. Indian J Orthop 44, 369-377, doi:10.4103/0019-5413.69305 (2010). [00319] P9 Rocchi, A. et al. CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis. J Clin Invest 120, 668-680, doi:10.1172/JCI36667 (2010). [00320] P10 Bernstein, M. et al. Ewing's sarcoma family of tumors: current management. Oncologist 11, 503-519, doi:10.1634/theoncologist.11-5-503 (2006). [00321] P11 Cerisano, V. et al. Molecular mechanisms of CD99-induced caspase- independent cell death and cell-cell adhesion in Ewing's sarcoma cells: actin and zyxin as key intracellular mediators. Oncogene 23, 5664-5674, doi:10.1038/sj.onc.1207741 (2004). [00322] P12 Kovar, H. et al. Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor. Oncogene 5, 1067-1070 (1990). [00323] P13 Weidner, N. & Tjoe, J. Immunohistochemical profile of monoclonal antibody O13: antibody that recognizes glycoprotein p30/32MIC2 and is useful in diagnosing Ewing's sarcoma and peripheral neuroepithelioma. Am J Surg Pathol 18, 486-494 (1994). [00324] P14 Parija, T. et al. Type 1 (11;22)(q24:q12) translocation is common in Ewing's sarcoma/peripheral neuroectodermal tumour in south Indian patients. J Biosci 30, 371-376 (2005). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00325] P15 Vural, C., Uluoglu, O., Akyurek, N., Oguz, A. & Karadeniz, C. The evaluation of CD99 immunoreactivity and EWS/FLI1 translocation by fluorescence in situ hybridization in central PNETs and Ewing's sarcoma family of tumors. Pathol Oncol Res 17, 619-625, doi:10.1007/s12253-010-9358-3 (2011). [00326] P16 Franzetti, G. A. et al. MiR-30a-5p connects EWS-FLI1 and CD99, two major therapeutic targets in Ewing tumor. Oncogene 32, 3915-3921, doi:10.1038/onc.2012.403 (2013). [00327] P17 Ventura, S. et al. CD99 regulates neural differentiation of Ewing sarcoma cells through miR-34a-Notch-mediated control of NF-kappaB signaling. Oncogene 35, 3944-3954, doi:10.1038/onc.2015.463 (2016). [00328] P18 Hingorani, P. et al. Increased CTLA-4(+) T cells and an increased ratio of monocytes with loss of class II (CD14(+) HLA-DR(lo/neg)) found in aggressive pediatric sarcoma patients. J Immunother Cancer 3, 35, doi:10.1186/s40425-015-0082-0 (2015). [00329] P19 Fujiwara, T. et al. Macrophage infiltration predicts a poor prognosis for human ewing sarcoma. Am J Pathol 179, 1157-1170, doi:10.1016/j.ajpath.2011.05.034 (2011). [00330] P20 Stahl, D., Gentles, A. J., Thiele, R. & Gutgemann, I. Prognostic profiling of the immune cell microenvironment in Ewing s Sarcoma Family of Tumors. Oncoimmunology 8, e1674113, doi:10.1080/2162402X.2019.1674113 (2019). [00331] P21 Guerzoni, C. et al. CD99 triggering in Ewing sarcoma delivers a lethal signal through p53 pathway reactivation and cooperates with doxorubicin. Clin Cancer Res 21, 146-156, doi:10.1158/1078-0432.CCR-14-0492 (2015). [00332] P22 Manara, M. C. et al. CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling. Oncotarget 7, 79925-79942, doi:10.18632/oncotarget.13160 (2016). [00333] P23 Menck, K. et al. Isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags. J Vis Exp, e51554, doi:10.3791/51554 (2014). [00334] P24 Fournier, N. et al. FDF03, a novel inhibitory receptor of the immunoglobulin superfamily, is expressed by human dendritic and myeloid cells. J Immunol 165, 1197-1209 (2000). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00335] P25 Coughlan, A. M. et al. Myeloid Engraftment in Humanized Mice: Impact of Granulocyte-Colony Stimulating Factor Treatment and Transgenic Mouse Strain. Stem Cells Dev 25, 530-541, doi:10.1089/scd.2015.0289 (2016). [00336] P26 Wunderlich, M. et al. AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3. Leukemia 24, 1785-1788, doi:10.1038/leu.2010.158 (2010). [00337] P27 Wang, J., Shiratori, I., Uehori, J., Ikawa, M. & Arase, H. Neutrophil infiltration during inflammation is regulated by PILRalpha via modulation of integrin activation. Nat Immunol 14, 34-40, doi:10.1038/ni.2456 (2013). [00338] P28 Shiratori, I., Ogasawara, K., Saito, T., Lanier, L. L. & Arase, H. Activation of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor. J Exp Med 199, 525-533, doi:10.1084/jem.20031885 (2004). [00339] P29 Lee, K. J. et al. Paired Ig-Like Type 2 Receptor-Derived Agonist Ligands Ameliorate Inflammatory Reactions by Downregulating beta1 Integrin Activity. Mol Cells 39, 557-565, doi:10.14348/molcells.2016.0079 (2016). [00340] P30 Kohyama, M. et al. Monocyte infiltration into obese and fibrilized tissues is regulated by PILRalpha. Eur J Immunol 46, 1214-1223, doi:10.1002/eji.201545897 (2016). [00341] P31 Weiskopf, K. Cancer immunotherapy targeting the CD47/SIRPalpha axis. Eur J Cancer 76, 100-109, doi:10.1016/j.ejca.2017.02.013 (2017). [00342] P32 Crompton, B. D. et al. The genomic landscape of pediatric Ewing sarcoma. Cancer Discov 4, 1326-1341, doi:10.1158/2159-8290.CD-13-1037 (2014). [00343] P33 Cironi, L. et al. IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells. PLoS One 3, e2634, doi:10.1371/journal.pone.0002634 (2008). [00344] P34 Yeap, W. H. et al. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes. Sci Rep 6, 34310, doi:10.1038/srep34310 (2016). [00345] P35 Sun, Y. et al. Evolutionarily conserved paired immunoglobulin-like receptor alpha (PILRalpha) domain mediates its interaction with diverse sialylated ligands. J Biol Chem 287, 15837-15850, doi:10.1074/jbc.M111.286633 (2012). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 EXAMPLE 6 [00346] Anti-CD99 Antibody Therapy Triggers Macrophage-Dependent Ewing Cell Death In Vitro and Myeloid Cell Recruitment In Vivo [00347] Abstract [00348] Background: Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, or refractory settings, we explored the downstream immune implications of targeting CD99. Methods: We discovered a human anti‐CD99 antibody (NOA2) by phagemid panning and investigated NOA2 immune‐cell mediated cytotoxicity in vitro and in vivo focusing on the myeloid cell compartment, given that M2 macrophages are present in human tumors and associated with a poor prognosis. Results: NOA2 is capable of inducing immune effector cell‐mediated Ewing death in vitro via engagement of macrophages. Mice with metastatic Ewing tumors, treated with NOA2, experience tumor growth arrest and an associated increase in intratumoral macrophages. Further, incubation of macrophages and Ewing cells with NOA2, in conjunction with anti‐PILRα antibody blockade in vitro, results in reactivation of previously dormant macrophages due to interrupted binding of Ewing CD99 to macrophage PILRα. Conclusions: These studies are the first to demonstrate the role of human immune effector cells in anti‐CD99 mediated Ewing tumor death. Engagement of CD99 by NOA2 results in recruitment of intratumoral macrophages. In addition, interruption of the CD99:PILRα checkpoint axis can be a relevant therapeutic approach to activate tumor‐associated macrophages. [00349] Introduction [00350] Ewing sarcoma is a rare tumor of the bone or soft tissues affecting approximately 250 children, adolescents, and young adults each year in the United States.1 Patients with chemotherapy‐responsive, localized disease have an excellent 5‐year overall survival of greater than 75%.2 Conversely, the presence of metastatic disease confers a very poor prognosis; less than 20% of these patients are cured.3,4 The mainstay of therapy for Ewing sarcoma consists of systemic chemotherapy, surgery and/or radiotherapy. While effective for the cohort of patients with localized disease, these treatment modalities confer the life‐long risk of organ toxicity, infertility, secondary malignancy, or disfigurement impacting quality of life.5,6,7 For the subset of patients with metastatic, recurrent, or DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 chemotherapy‐refractory disease, overall survival is dismal and little progress has been made in decades.8 [00351] Ewing sarcoma is characterized histologically by small, round blue cells and a diffuse membranous immunohistochemical stain for CD99 (or MIC2), a 32kDa type I membrane glycoprotein.9‐13 Eighty‐five percent or more of Ewing sarcoma cases harbor a chimeric fusion between the RNA binding protein EWS and an ETS family transcription factor (i.e., EWS‐FLI1) creating a fusion oncogene (t(11;22)(q24;q12)) deemed relevant to tumor pathogenesis.14,15 While the published literature indicates an important link between CD99 and EWS‐FLI1, this relationship is incompletely characterized.16,17 In addition, the Ewing immune landscape has traditionally been described as a “cold” immune state. However, the presence of CD68+ tumor‐associated macrophages (TAMs) has been linked to poor clinical outcomes.18‐20 [00352] In the absence of available EWS‐FLI1 targeted agents, therapeutic antibodies targeting CD99 have been investigated. Anti‐CD99 antibody blockade has been shown to induce Ewing cell aggregation and caspase‐independent apoptosis11, trigger Ewing cell death in p53 wild‐type patient‐derived cell lines21, and more recently incite cell death by a process termed methuosis, a form of non‐apoptotic cell death characterized by displacement of the cytosol by vacuoles derived from macropinosomes, involving the IGF1R/Ras/Rac1 signaling pathway.22 However, the role of the human immune system in anti‐CD99 mediated Ewing cell death has not yet been explored. [00353] In this study we discovered a high‐affinity human anti‐CD99 antibody, NOA2, capable of binding Ewing sarcoma cells, and subsequently explored the in vitro and in vivo activity of this antibody to engage macrophages, impact tumor growth, and modulate macrophage function. NOA2 can induce Ewing cell death through engagement of macrophages and antibody‐mediated cellular phagocytosis in vitro. Tumors from humanized xenograft mice treated with NOA2 undergo growth arrest and contain intratumoral infiltrates of human macrophages. Furthermore, inhibition of the binding of Ewing CD99 to macrophage PILRα, an inhibitory receptor and CD99 ligand, by dual anti‐CD99 and anti‐ PILRα blockade leads to macrophage reactivation. Single blockade of target does not achieve this endpoint. Our results indicate that the binding of CD99 by NOA2 and disruption of the CD99:PILRα can modulate myeloid cell activity. [00354] Materials and Methods DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00355] Cell Culture [00356] CD99 positive, human Ewing sarcoma cell lines (A673, TC32, SKNEP‐1, TTC466, 79 RDES, TC71 and CADO‐ES1) were obtained and previously authenticated and tested negative for mycoplasma. CD99 negative Kelly neuroblastoma cells were also obtained. A673, SKNEP‐1, and Kelly cells were cultured in DMEM (Gibco) and TC32, TTC466, RDES, TC71, and CADO‐ES1 were cultured in RPMI (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin‐streptomycin (Cellgro). Cells were incubated at 37ºC with 5% CO2.293F cells were purchased from Thermo Fisher and cultured in Freestyle medium (Gibco). Cells were passaged a maximum of 8‐10 times over 4‐8 weeks. [00357] Flow cytometric characterization of anti-CD99 binding activity [00358] Ewing sarcoma cells of various lines were incubated in a florescence‐activated cell sorting (FACS) tube with 2μg/mL of each anti‐CD99 monoclonal antibody for one hour at 4ºC followed by staining with a FITC‐conjugated mouse anti‐human IgG. Titration analyses were subsequently performed with NOA2 given that this candidate antibody demonstrated the strongest binding across Ewing lines. CD99 negative Kelly neuroblastoma cells were used as a negative control. Kinetic interactions between anti‐CD99 antibody clones and soluble CD99‐Fc were measured utilizing the Octet RED system. NOA2 and NOA3 were digested to form F(ab’)2 fragments such that the soluble CD99‐Fc can be immobilized on human Fc probes as the ligand while the F(ab’)2 fragments, at concentrates ranging from 0nM to 100100nM, can serve as the analyte. ForteBio Octet software was utilized to analyze the data. Once binding curves were established, the experiment was repeated using analyte concentrations in a narrow range (10‐15 nm for NOA2 and 10‐25 nm for NOA3) so as to more accurately calculate each antibody KD. The following settings were utilized: Baseline 60 seconds (s) at 1000rpm, Loading 300 s at 1000rpm, Association 300 s at 1000rpm, and Dissociation 600 s at 1000rpm. [00359] Human effector cell assays [00360] Antibody‐dependent cell‐mediated cytotoxicity (ADCC) assays were performed with the use of an ADCC Reporter Bioassay Core Kit (Promega #G7010, G7018). Serial dilutions of NOA2 or an isotype control were plated with Ewing cells, or CD99 negative Kelly neuroblastoma cells, at concentrations ranging from 0 μg/mL to 2 μg/mL. Effector cells were plated at an effector‐to‐target cell ratio of 2.5:1 and incubated for 6 hours at 37ºC with 5% CO2. A Bio‐Glo Luciferase Assay was utilized to quantitate cell death. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 Plates were read on a POLARStar Omega plate reader and relative light units (RLU) analyzed for each condition after subtracting for background. ADCC was performed in duplicate for each cell line. Error bars denote standard deviation. [00361] Antibody‐dependent cellular phagocytosis (ADCP) assays were performed by first isolating human monocytes from human peripheral blood mononuclear cells (PBMCs). PBMCs were plated in MDM medium (DMEM with 10% heat‐inactivated human serum, 1% penicillin/streptomycin, 1% L‐glutamine, and 50ng/mL GM‐CSF) in a Petri dish for 3 days at 37ºC with 5% CO2. At the end of three days, the supernatant was removed and MDM media replenished to further culture the adherent macrophage precursors. On day 7, macrophages were incubated with 0.5mM EDTA in PBS, lifted, and added at a ratio of 1:4 (target: effector cell) to Ewing sarcoma cells stained with PKH26 fluorescent membrane dye (Sigma Aldrich). Isotype IgG or NOA2 was added to the wells and incubated at 37ºC for 4 hours. After co‐ culture, cells were imaged using a Celigo imaging cytometer (Nexcelom Biosciences) and then lifted and transferred to a 96‐well V‐bottom plate for FACS. Cells were then counterstained with FITC‐anti‐CD14 for 30 minutes on ice. Engulfed target cells were defined as PKH26+/CD14+. ADCP was performed in duplicate for each cell line. Statistics were calculated using a two‐tailed students t‐test with a p value <0.05 denoting significance. [00362] In vivo cytotoxicity assays [00363] An in vivo experiment was first performed utilizing male and female NOD scid gamma (NSG) mice injected via tail vein with 20,000 TC32 Ewing sarcoma cells transduced with a Luc‐mCh vector to allow for bioluminescence imaging (BLI). Mice were approximately 4‐6 weeks in age and weighed approximately 25 g. BLI was performed weekly (Xenogen IVIS) until tumor burden (liver micro‐metastases) can be reliably quantitated after 5 weeks of growth.1 x 107 human peripheral blood mononuclear cells (PBMCs) were injected via tail vein at 5 weeks and mice were treated twice weekly with 5mg/kg of isotype control (n= 2 mice) or NOA2 (n=3) with the first treatment initiated 24 hours following PBMC infusion. BLI was performed weekly and regions of interest (ROIs) drawn over the liver to quantify serial mean luminescence (in units of total flux (p/s)). Mice were euthanized after 4 treatments, prior to the development of graft versus host disease, and tumor tissue harvested for hematoxylin eosin staining and immunohistochemistry. Immunohistochemistry slides were imaged on a confocal microscope using a Nikon camera or the Keyence BZ‐X800 system (Keyence Co. Itasca, IL). Cells of various immune subtypes were counted from three 10x high‐powered fields in representative tumors from mice treated with IgG or NOA2 and a DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 student’s two‐tailed t‐test (with significance denoted by a p‐value <0.05) run to compare counts. Error bars are included to denote standard deviation. [00364] Flow cytometry of CD45+ cells isolated from subcutaneous Ewing tumors [00365] A follow‐up in vivo experiment, with endpoint analyses to be reported elsewhere, established subcutaneous Ewing sarcoma tumors in humanized mice. Neonatal NSG‐SGM3 mice underwent whole‐body irradiation (100cGy) and tail vein injection of 2.5 x 105 CD34+ hematopoietic stem cells (HSCs). After 10‐12 weeks, flow cytometric analysis of circulating blood was performed to confirm engraftment defined as >20% peripheral blood human CD45+ cells. Subcutaneous flank tumors were established in 30 mice after injection of 1x106 luc‐mCh transduced TC32 cells. Animals were randomized to treatment with 3mg/kg of isotype IgG (n=12) versus NOA2 (n=15) for a total of 6 treatments delivered over three weeks; treatment began when tumors measured approximately 150‐200 mm3. Disease remained non‐metastatic and confined to the subcutaneous region. Two mice at baseline and one mouse from each treatment cohort were sacrificed after 2, 4 and 5 treatments. Tumors from these timepoints were dissociated to a single‐cell suspension, Ficoll’ed to isolate the buffy coat, and prepared for flow cytometry. Cells were gated for human CD45+ expression and the percentage of human CD33+ cells, as a subset of CD45+ cells, was reported. For statistical analysis, tumors from each treatment cohort (n=3 treated with IgG, n=3 treated with NOA2) were grouped and a student’s two‐tailed t‐test performed with significance denoted by a p‐value <0.05. Error bars are included to denote standard deviation. [00366] IGF-1 as a macrophage chemoattractant [00367] Peripheral blood monocytes (PMNs) were isolated from human PBMCs by double gradient centrifugation.23 Following isolation, monocytes were diluted to a concentration of 3 x106 cells/ml in complete RPMI‐1640 (Thermo Fisher), supplemented with 10% FBS, 100U/ml Penicillin/Streptomycin, 4.5g/L D‐glucose, 2.383g/L HEPES, L‐ Glutamine, 15g/L sodium bicarbonate, and 110mg/L sodium pyruvate.3 x 104 cells were inserted in the upper chamber of a 96‐well TC‐treated HTS Transwell Plate (Corning, CLS3388) with a 5.0μm pore polycarbonate membrane. A chemotactic gradient was established using different concentrations of MCP‐1 (monocyte chemoattractant protein‐1) or IGF‐1 (0.5 to 100ng/mL) in media supplemented with 8μM Hoechst dye (Thermo Fisher). After 4 hours, the inserts were removed. Cells were spun down and the total fluorescent count from each well, as a reflection of cell migration, was recorded using a Celigo Imaging DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 Cytometer (Nexcelom Bioscience, LLC). These experiments were performed in triplicate and statistical significance calculated using a paired two‐tailed student’s t‐test with significance denoted by a p‐value <0.05. Error bars are included to denote standard deviation. [00368] PILRα synthesis and analysis of binding to human CD99 [00369] Human His‐tagged PILRα was synthesized by cloning the extracellular domain into a pcDNA3.4 vector and transfecting Expi293F cells (Life Technologies). Protein was isolated from the supernatant and purified utilizing a HisPur Ni‐NTA column (Qiagen). An SDS page gel demonstrated a band at 55kDa. Protein synthesis was subsequently confirmed by ELISA, coating a plate with 2 μg/mL of our synthesized PILRα protein and incubating with a commercially available anti‐human PILRα antibody (Sigma Aldrich) at a concentration of 2 μg/mL to assure binding. After verification, binding of human PILRα to human CD99 was assessed by ELISA. An ELISA plate was coated with 2 μg/mL of human CD99 and incubated with PILRα at concentrations ranging from 0.25 to 5 μg/mL and an anti‐ PILRα antibody with HRP‐bound secondary. [00370] Macrophage function in the context of PILRα and CD99 blockade [00371] Monocyte‐derived macrophages were isolated from PBMCs and identified as the cells left adherent following 5‐7 days of PBMC culture. They were primed with 50ng/mL IFNγ (Novus Biologicals) and triggered for M1 polarization with 10ng/mL lipopolysaccharide (LPS, Sigma Aldrich).24 Twenty‐four hours after LPS treatment, control group macrophages were incubated with one of three antibody conditions: anti‐CD99 alone, anti‐PILRα alone, or the combination of both antibodies. The experimental group macrophages were co‐incubated with CD99+ TC32 Ewing cells at a ratio of 2:1 (macrophage:Ewing cell) or Kelly CD99‐ neuroblastoma cells and subsequently treated with each of the three antibody conditions. This experiment was performed with Ewing cells on two separate occasions using different macrophage human donors and in duplicate, using a third donor, with Kelly neuroblastoma cells. Twelve hours later the supernatant was collected. ELISA was performed on the supernatant to assess changes in the concentration of TNF‐α as a marker of macrophage activation (Abcam). [00372] Phagemid panning for identification of anti-CD99 scFv’s [00373] Phagemid panning, utilizing a 27 billion member human single-chain variable fragment (scFv)-phage library, was performed for three rounds against immunotube-bound human CD99-Fc (G&P Biosciences). NUNC immunotubes were coated with soluble CD99 DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 (10μg/mL) and incubated at 4°C overnight. They were subsequently washed with phosphate- buffered saline (PBS) and blocked with 4% milk/PBS at 37°C for 1-2 hours. During blocking, TG1 bacteria were grown in 2xYT media in a baked flask at 37°C, shaking at 250 rpm until growth was recorded at an OD600 of 0.5-0.7. The phagemid library was prepared for panning by diluting 5 x 1012 particles/mL in 2% milk/PBST (PBS-Tween), supplementing with isotype IgG to mitigate against binding to the Ig portion of CD99 Fc. Following immunotube blocking, tubes were washed with PBS, loaded with 1 mL of the phage sample, and incubated for 90 minutes on a rotating platform and then stationary, for 30 minutes, at room temperature. Immunotubes were washed and bound-phage eluted using triethylamine. [00374] Eluted phage were neutralized with 1M Tris and used to infect TG1 cells. Serial dilutions of infected bacterial cultures were plated and grown overnight at 37°C, while the remainder of the culture was plated on a 15 cm bacterial dish and grown overnight at 30°C. The next morning, bacteria were harvested from the 15 cm dish and grown until an OD of 0.5-0.7 was achieved. A volume of 10 mL of this culture was infected with VCS-M13 helper phage at a multiplicity of infection (MOI) of 20. Bacterial cells were resuspended in 2xYT containing ampicillin and kanamycin, grown overnight, and centrifuged the following morning. Supernatant was collected and combined with 20% PEG/2.5 M NaCl in a 4:1 ratio, incubated on ice for 1 hour, and centrifuged. The resultant phage pellet was resuspended at 2mL and stored at 4°C. These rescued phage were then utilized for subsequent panning rounds against diminishing concentrations (lowest 1μg/mL) of immunotubebound CD99. [00375] Individual colonies from each round of panning were picked, cultured and analyzed for CD99 binding by ELISA. Positive binders were sequenced to determine the number of unique scFv binding clones. Each unique clone was assessed for binding to membrane-associated CD99 on patient-derived Ewing sarcoma cell lines by fluorescence- activated cell sorting (FACs). The resultant cell-binding scFv’s were screened by ELISA against serial dilutions of soluble CD99 to identify the top three clones (NOA1, 2, and 3) with reproducible differential binding at concentrations of 0.5 μg/mL or less. [00376] Molecular cloning and expression of anti-CD99 mAbs [00377] The anti-CD99 heavy chain domains for each scFv were amplified and cloned into the MluI and NheI sites of a TCAE6-LL2 IgG expression vector, while scFv lambda light chains were cloned into the AvrII and HindIII sites. The final plasmids were DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 transformed into E. Coli (strain DH5α). DNA was extracted from selected clones and sequenced to verify appropriate digestion and ligation.293F cells were transfected with the anti-CD99 scFvFc expression plasmids and the cell culture supernatant was collected at day 7, filtered through a 45μm filter, and purified by Protein A affinity chromatography. SDS- PAGE gels were run to assess protein quality. [00378] Primers utilized for molecular cloning [00379] The following forward primers were used for heavy chain cloning: clones NOA1 and NOA2 forward: 5’- ATC GAC GCG TGT CCT GAG CCA GGT GCA GCT GGT G-3’ and NOA3 forward: 5’ - ATC GAC GCG TGT CCT GAG CGA GGT GCA GCT GGT G-3’. The following reverse primers were used for heavy chain cloning: clones NOA1 and NOA2 reverse: 5’ – CAT GCG CTA GCT GAA GAG ACG GTG ACC AT-3’ and clone NOA3 reverse: 5’ – CAT GCG CTA GCT GAG GAG ACG GTG ACC GT-3’. The following forward primers were used for light chain cloning: clone NOA1 forward: 5’ – ATC CCA AGC TTA AGC CAG TCT GTG CTG AC T-3’; clone NOA2 forward: 5’ – ATC CCA AGC TTA AGC CTG CCT GTG CTG ACT-3’; and clone NOA3 forward: 5’ – ATC CCA AGC TTA AGC TCC TAT GAG CTG ACT-3’. The reverse primers used for light chain cloning were as follows: clone NOA1 reverse: 5’- GCT GAC CTA GGA GGA CGG TGA CCT T-3’ and clones NOA2 and NOA3 reverse: 5’- GCT GAC CTA GGA GGA CGG TCA GCT T- 3’. [00380] Flow cytometric analysis of CD47 expression [00381] CD47 Ewing cell surface expression was analyzed by FACs for each Ewing sarcoma cell line at baseline and following treatment with NOA2 antibody. Ewing sarcoma cells of various lines (A673, TC32, CADO-ES1) were incubated in a FACs tube alone or with varying concentrations of NOA2 antibody (range: 0.125 to 2.0 μg/mL) for one hour and secondarily stained with 1.0 μg/mL of anti-CD47 FITC bound antibody (ThermoFisher Scientific). Percentage of CD47-expressing cells, as a function of pre-incubation NOA2 concentration, was tabulated. [00382] Flow cytometric analysis of CD99 expression on PBMCs [00383] A total of 1 x 106 unstained peripheral blood mononuclear cells were stained first with 2 μg/mL of NOA2 for one hour on ice, washed with autoMACs rinsing buffer (Militenyi Biotec) containing 1% BSA, and then incubated with 0.02 μL/mL of anti-human FITC-bound secondary (BD pharmingen, per manufacturers specifications). Cells were then DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 washed and subsequently incubated with 0.02 μL/mL APC-bound anti-human CD33 (Biolegend, per manufacturers specifications). Cells were again washed three times in autoMACs rinsing buffer with 1% BSA and prepared for FACS. [00384] Kinetic analysis of binding interactions between anti-CD99 and soluble CD99 [00385] Kinetic interactions between anti-CD99 antibody clones and soluble CD99-Fc (G&P Biosciences) were measured utilizing the Octet RED system (ForteBio). NOA2 and NOA3 were digested to form F(ab’)2 fragments such that the soluble CD99-Fc can be immobilized on human Fc probes as the ligand, while the F(ab’)2 fragments, at concentrates ranging from 0nM to 100nM, can serve as the analyte. NOA1 was omitted from this assay due to low-affinity binding across Ewing lines by FACs. ForteBio Octet software was utilized to analyze the data. Once binding curves were established, the experiment was repeated using analyte concentrations in a narrow range (10-15 nm for NOA2 and 10-25 nm for NOA3) so as to more accurately calculate each antibody KD. The following settings were utilized: Baseline 60 seconds (s) at 1000rpm, Loading 300 s at 1000rpm, Association 300 s at 1000rpm, and Dissociation 600 s at 1000rpm. [00386] Results [00387] Binding of anti-CD99 antibodies to Ewing sarcoma cells by flow cytometry [00388] The binding of anti‐CD99 mAbs to CD99 was first examined by FACS. Three anti‐CD99 antibodies (NOA1, 2, and 3) were isolated by phage display and characterized (Table 12). FACS binding of IgG1 isoforms to CD99‐positive patient‐derived Ewing sarcoma cell lines was performed (FIG.24). NOA1 (blue) demonstrated limited binding at concentrations as high as 10 μg/mL whereas NOA2 (red) and 3 (green) demonstrated strong binding at concentrations of 2 μg/mL (FIG.24A (left)). None of the clones bound to CD99‐ negative Kelly neuroblastoma cells (FIG.24A (right)). NOA2 was carried forth for further studies given superior binding across Ewing cell lines. The relative affinity of NOA2 was determined by flow cytometric saturation binding studies against five independent CD99‐ positive Ewing sarcoma cell lines (FIG.24B). NOA2 binding was specific for CD99‐positive Ewing cells and demonstrated circa 3‐fold range in binding affinity across five Ewing lines. OctetRed was performed and revealed a Kd 3.35 x 10‐9 M +/‐ 4.60 x 10‐11 M with Kon 4.15 x 105 +/‐ 4.04 x 103 (1/Ms) and Koff of 1.39 x 10‐3 +/‐ 1.35 x 10‐5 (1/s) (FIG.24C). FIG.24D demonstrates the heavy and light chain sequences and comparative human germlines for the NOA2 scFv. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00389] Anti-CD99 antibodies mediate killing of CD99+ Ewing sarcoma cells by ADCC and ADCP [00390] To determine if immune effector cells play a role in NOA2 antibody‐mediated cytotoxicity, ADCC and ADCP were performed. NOA2 triggered Ewing cell death as a function of antibody concentration in ADCC assays, however the magnitude of cell killing was cell line specific. As shown in FIG.25A‐C, Ewing cell death as assessed by net luminescence was more pronounced for CADO‐ES1 and TC32. Ewing cells retained viability when incubated with an isotype control and CD99‐negative Kelly neuroblastoma cells. [00391] For in vitro ADCP studies, macrophages isolated from human PBMCs and co‐ incubated with Ewing sarcoma cells and NOA2 showed aggregation and engulfment of tumor cells (FIG.25D, arrows). Similar to ADCC, these ADCP results were more pronounced for CADO‐ES1 and TC32. FACS demonstrated an increase in PKH26+/CD14+ cells, representing Ewing cells engulfed by macrophages following NOA2 incubation; the two NOA2 concentrations tested for ADCP (2 & 5 μg/ml) did not show an antibody dose‐ dependent effect (FIG.25E-F). ADCP was most prominent visually following incubation with 2 μg/mL indicating that lower concentrations of NOA2 can be more effective for this immune effector function. The lack of dose‐dependent effect on the percent of FITC+/PE+ cells on flow cytometry can be secondary to an increase in cell death or clumping at the higher antibody concentration. There was comparatively minimal aggregation and engulfment of Ewing sarcoma cells with incubation of isotype IgG. In summary, NOA2 kills Ewing cells by both ADCC and ADCP, with differing efficiency across cell lines, implicating immune cells in antibody‐mediated cytotoxicity. [00392] In vivo Ewing tumor growth arrest following treatment with NOA2 and resulting myeloid cell infiltrate [00393] We next sought to determine whether anti‐CD99 antibody treatment of mice harboring micro‐metastatic Ewing sarcoma tumors can inhibit tumor growth. An in vivo experiment was performed with a human peripheral blood leukocyte (PBL)‐NSG humanized mouse harboring Ewing sarcoma liver micro‐metastases. Mice were treated with NOA2 (n=3) or an isotype control (n=2), initiated 24 hours after PBMC injection, for a total of 4 treatments (arrows). FIG.26A demonstrates tumor growth arrest at 14 days post-treatment, as extrapolated by mean luminescence, in mice treated with NOA2 as compared with an isotype control. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 [00394] To investigate NOA2’s mode of action, mice were euthanized following therapy and livers dissected, embedded in paraffin, and sectioned (FIG.26B). The representative images demonstrate a more prominent human CD45, mouse CD14, and human CD33 and CD16 infiltrate. There was a statistically significant increase in human CD45+, mouse CD14+, and human CD33+ infiltrating cells (p<0.05) in mice treated with NOA2 compared to IgG when averaged over three 10x high‐powered fields. The differences for human CD16+ cells (p<0.16) did not reach statistical significance, due to small tumors and low overall cell counts (FIG.26C). In NOA2 treated mice there was also a noticeable difference in the appearance of mouse myeloid cells staining for CD14; these cells were more plump and aggregated than in IgG‐treated tumors indicating macrophage activation, similar to that seen in in vitro ADCP studies. Anti‐CD99 treated tumors also demonstrated pockets of necrosis indicative of apoptosis by TUNEL staining (FIG.26B). [00395] The observation that treatment with NOA2 led to an accumulation of hCD45+ and hCD33+ immune cells in Ewing tumors in the PBL‐NSG mouse study described herein led us to perform a second treatment study in CD34+ HSC reconstituted NSG‐SGM3 mice which show enhanced engraftment of human myeloid cells.25,26 Mice were treated twice weekly with IgG or NOA2 and subcutaneous tumors harvested from mice at baseline and from each cohort following treatments 2, 4 and 5. Human CD45+ cells were isolated from the harvested subcutaneous tumors and analyzed by FACS. Cells were gated for human CD45+ expression; the percentage of CD45+ cells expressing human CD33+ at baseline compared to the three timepoints is shown in FIG.26D. Tumors from the isotype or NOA2 treatment cohorts were grouped to allow for statistical analyses. As shown in FIG.26E there is a robust human CD33+ myeloid cell infiltrate in NOA2‐treated mice with ~55% of hCD45+ cells being derived from the myeloid lineage (p<0.01). In summary, these collective results demonstrate that NOA2 treatment of humanized mice bearing Ewing tumors leads to the recruitment of CD33+ monocytes/myeloid cells. [00396] IGF-1 mediated macrophage chemotaxis [00397] Given prior reports documenting upregulation of the IGF/IGF‐1R axis with Ras/Rac1 signaling pathway in Ewing sarcoma, we performed immunohistochemistry on tumors from the micro‐metastatic in vivo experiment. FIG.27A demonstrates more prominent staining for IGF‐1R, Ras, and IGF‐1 in tumors from mice treated with NOA2 as compared with IgG. We sought to determine whether IGF‐1 was contributing to recruitment of blood monocytes to the tumors. Monocyte trafficking was analyzed in a transwell assay DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 utilizing a chemotactic gradient of MCP‐1 (positive control, FIG.27B left) or IGF‐1 (FIG. 27B right). A statistically significant increase in monocyte chemotaxis, compared with control, was observed with IGF‐1 at doses greater than 10 ng/mL. [00398] Macrophage activation following co-incubation of Ewing sarcoma cells with anti-CD99 and anti-PILRα antibodies [00399] PILRα is a CD99 ligand, and inhibitory receptor, that is preferentially expressed on human myeloid cells but also on natural killer cells, dendritic cells, and other granulocytes. PILRα has been implicated in and can be restrictive of immune cell recruitment and activation.27‐30 Mouse PILRα has been shown to bind mouse CD99 but this protein interaction has not been well defined in humans.31 As shown in FIG.28A we confirmed dose‐dependent binding of human PILRα to soluble human CD99 by ELISA. [00400] With the knowledge that human macrophages express PILRα24 and confirmation that human PILRα binds human CD99, we next sought to evaluate the effects of blocking the macrophage PILRα:CD99 Ewing sarcoma binding axis (FIG.28B, left panel) on macrophage activity using TNF‐α secretion as a proxy. In addition to expressing PILRα, macrophages, like Ewing cells, also express CD99 which can be bound by NOA2. As shown in FIG.28C (upper and lower panels) for three donors, TNF‐α secretion decreases when M1 macrophages (dark bars) are incubated with the anti‐CD99/anti‐PILRα cocktail but not with each antibody alone, indicating that an inhibitory axis is triggered by the binding of both macrophage surface proteins (FIG.28B, right upper panel). This effect persists when M1 macrophages are incubated with CD99 negative Kelly cells (FIG.28C, lower). However, when the M1 macrophages are incubated with CD99 positive TC32 cells, interrupting the binding of the PILRα:CD99 axis ((FIG.28B, right upper panels) TNF‐α secretion is restored perhaps due to saturation of anti‐CD99 on Ewing cells diverting binding from M1 macrophages. Further mechanistic studies will be required to determine this mode of action and analyze the anti‐tumor outcome of this combination immunotherapy in vivo. [00401] Isolation of CD99-specific scFv’s from a phage display library and their genetic analysis [00402] Two non-immune human scFv-phage display libraries containing 12 (Mehta I) and 15 (Mehta II) billion members were combined and incubated with immunotube-bound CD99-Fc. Bound scFvs were eluted from each panning round and screened by ELISA to confirm binding specificity. ELISA was first performed against an isotype-matched human DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 monoclonal IgG1 to subtract out Fc-region binders, and subsequently against soluble CD99- Fc. Of the 50 resultant clones, DNA sequence analysis revealed that 10 were unique. Six of these phagemid scFvs bound to A673 Ewing sarcoma cells by FACs. Binding affinity for these six scFv’s was assessed by ELISA with titration of soluble CD99-Fc concentrations. The top three clones, NOA1, 2, and 3 demonstrated saturable binding at concentrations of 0.125μg/mL or less. These scFvs were later used for further studies. [00403] The germline Ig gene segment usage and somatic hypermutation (SHM) are shown in Table 12. The NOA2 heavy chain is most closely aligned with the *03 allele of the IGHV5-51 germline gene, the *01 allele of the IGHD1-26 germline gene, and the *02 allele of the IGHJ3 germline segment. The heavy chain harbors seven single-nucleotide mutations (SNMs) including CDR1; however, CDR2 is in the germline configuration. The light chain utilizes IGLV5-37*01 and IGLJ3*02 germline genes and has six SNM. [00404] Ewing cell CD47 expression following incubation with NOA2 [00405] We next investigated whether CD47, a surface molecule known to inhibit phagocytosis was involved in the differential ADCP effects among the cell lines described herein. FACS analysis was performed as a function of pre-treatment with various concentrations of NOA2. As can be seen in FIG.29, while lower concentrations of NOA2 consistently showed a decrease in CD47 expression in three cell lines, A673 cells showed circa 35% upregulation in expression at the highest concentrations of NOA2 compared to 18% and 0% increased in expression in CADO and TC32 cells. It is unclear if this modest increase in CD47 expression can account for the lack of ADCP in A673 cells. [00406] Monocyte expression of CD99 as detected by NOA2 [00407] PBMCs were gated by side and forward scatter to separate monocytes and lymphocytes by size. There was non-specific binding of anti-human FITC-bound secondary to monocytes which was accounted for by gating. Nearly 70% of CD33-positive monocytes demonstrated circa 3-fold binding of NOA2 (FIG.31). [00408] Discussion [00409] Here we report on the discovery of NOA2, a human monoclonal antibody that targets CD99 and is capable of recruiting and reactivating components of the innate immune system to direct antibody‐mediated Ewing sarcoma death. NOA2 can trigger antibody‐ mediated tumor death through ADCC (FcγR‐mediated killing) and ADCP (phagocytosis). In DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 vivo treatment of Ewing tumor‐bearing mice with NOA2 results in a tumoral infiltrate of morphologically activated mouse CD14+ cells and an increase in human CD33+ cells. NOA2 is also associated with upregulation of IGF‐1, potentially contributing to monocyte/macrophage chemotaxis and recruitment. Combination NOA2 and PILRα blockade in vitro potentially reverses the inhibitory CD99:PILRα pathway linking Ewing sarcoma cells and macrophages leading to restoration of TNF‐α secretion. In light of published data showing M2 tumor‐protective macrophages to be the predominant infiltrating immune subtype in human Ewing tumors, therapeutic mechanisms aimed at macrophage recruitment and activation are worthy of further study.19,20 Our studies focused predominantly on M1 macrophages, given recent data to indicate that ligation of CD99 and use of agents targeting the PI3/AKT pathway can promote an M1 phenotype. [00410] We investigated several immune‐mediated mechanisms of action to further understand the tumoricidal activities of NOA2. We observed the differential ability of NOA2 to induce ADCC and ADCP in CADO‐ES1 and TC32 cells as compared with A673 cells. A673 cells harbor a BRAFV600E mutation which is unusual in Ewing sarcoma and can have an atypical driver role besides the EWS fusion gene.32 We attempted to reconcile the differences in ADCP killing by examining the potential overexpression of CD47, recognizing that the CD47/SIRPα axis has been implicated in the control of myeloid cell activation.33 At baseline, human Ewing‐derived cell lines express CD47, however, we could not demonstrate a clear upregulation of CD47 on A673 cells following treatment with NOA2 at concentrations of 1μg/mL or higher (FIG.29). [00411] CD99 antibody blockade has been shown to trigger Ewing cell death through upregulation of the IGF‐1R/Ras/Rac1 signaling pathway and a process termed methosis.22 While our data corroborates upregulation of this pathway following binding of NOA2 therapy, NOA2 does not trigger Ewing cell death in the absence of immune cell engagement. This prompted us to evaluate whether the IGF‐1R/Ras/Rac1 signaling pathway serves a different purpose following NOA2 binding. The EWS‐FLI1 fusion protein has been shown to activate the IGF1 promotor and induce IGF‐1 expression.34 We hypothesize that NOA2 can serve as an agonist on Ewing cells, upregulating EWS‐FLI1 leading to enhanced IGF‐1 transcription (FIG.30). We have shown by immunohistochemistry that IGF‐1 is upregulated in the tumors of NOA2‐treated mice and that this protein can serve as a monocyte chemoattractant (FIG.27) explaining the more prominent myeloid infiltrate visualized in NOA2‐ vs, IgG‐treated tumors from our in vivo studies. Mouse CD14+ cells from our initial in vivo experiment appeared morphologically activated, similar to those seen in our ADCP DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 studies. On the basis of these findings, we postulate that NOA2 is responsible for recruiting activated, intratumoral macrophages which can contribute to early tumor growth arrest and apoptosis, as visualized by TUNEL staining.35 [00412] Our exploration of the PILRα:CD99 axis was an attempt to address whether macrophages can be re‐activated through disruption of PILRα:CD99 binding. Macrophages are known to express both surface PILRα and CD99 while Ewing cells express only CD99; our NOA2 antibody binds both macrophage CD99 (FIG.31) and Ewing CD99 while the mouse anti‐human PILRα antibody we utilized binds only macrophages. We show that human PILRα binds to human CD99 thereby indicating a mechanism by which human macrophages and Ewing cells can interact.31 When macrophages are incubated with both anti‐CD99 and anti‐PILRα antibodies, TNF‐α secretion decreases indicating that both of these cell surface proteins are implicated in macrophage activity. When macrophages are co‐ incubated with a CD99 negative Kelly neuroblastoma cell line, and incubated with both antibodies there is little change in TNF‐α decline. Incubation of M1 macrophages with Ewing CD99+ cells, conversely, introduces additional CD99 binding sites as well as a new Ewing:macrophage, CD99: PILRα, axis predicted to contribute to macrophage suppression. In this context, incubation with both anti‐CD99 and anti‐PILRα antibodies, interrupts the Ewing:macrophage axis and also diverts NOA2 binding to Ewing CD99 – the combination of which reverses macrophage suppression allowing a rebound in TNF‐α secretion. These experiments are limited by the number of macrophages isolated from each donor and donor variability in macrophage function. This data support additional exploration of this axis. [00413] Conclusion [00414] The study of tumor‐infiltrating lymphocytes has received considerable attention, given the striking successes in use of PD‐1 inhibitors for adult malignancies. However, these agents are less likely to be of benefit in Ewing sarcoma given that Ewing tumors are mutationally silent and harbor a paucity of T‐cells.18‐20,36 This is the first study to demonstrate the role of myeloid cells in antibody‐mediated Ewing cell death and to identify a new immune axis capable of reactivating tumor‐associated macrophages. Should anti‐CD99 therapeutics gain further traction for study in patients with Ewing sarcoma, important considerations for on‐target, off‐tumor toxicity will be relevant given expression of CD99 on other human tissues, most notably leukocytes and pancreatic beta‐islet cells. For the population of Ewing sarcoma patients with metastatic, relapsed, or refractory disease, there is a grave need for new therapeutics. For patients with single‐site disease cure still comes at the DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 expense of significant long‐term toxicities. Pivoting focus towards manipulation of the myeloid compartment can therefore be of therapeutic relevance for Ewing sarcoma and other mutationally silent tumors for which new therapies are sorely lacking. [00415] References [00416] 1. Gurney JG, Severson RK, Davis S, et al: Incidence of cancer in children in the United States. Sex‐, race‐, and 1‐year age‐specific rates by histologic type. Cancer 75:2186‐95, 1995 [00417] 2. Womer RB, West DC, Krailo MD, et al: Randomized controlled trial of interval‐compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Childrenʹs Oncology Group. J Clin Oncol 30:4148‐54, 2012 [00418] 3. Grier HE, Krailo MD, Tarbell NJ, et al: Addition of ifosfamide and etoposide to standard chemotherapy for Ewingʹs sarcoma and primitive neuroectodermal tumor of bone. N Engl J Med 348:694‐701, 2003 [00419] 4. 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Sun Y, Senger K, Baginski TK, et al: Evolutionarily conserved paired immunoglobulin‐like receptor alpha (PILRalpha) domain mediates its interaction with diverse sialylated ligands. J Biol Chem 287:15837‐50, 2012 [00447] 32. Gouravan S, Meza‐Zepeda LA, Myklebost O, et al: Preclinical Evaluation of Vemurafenib as Therapy for BRAF(V600E) Mutated Sarcomas. Int J Mol Sci 19, 2018 [00448] 33. Weiskopf K: Cancer immunotherapy targeting the CD47/SIRPalpha axis. Eur J Cancer 76:100‐109, 2017 [00449] 34. Cironi L, Riggi N, Provero P, et al: IGF1 is a common target gene of Ewingʹs sarcoma fusion proteins in mesenchymal progenitor cells. PLoS One 3:e2634, 2008 [00450] 35. Yeap WH, Wong KL, Shimasaki N, et al: CD16 is indispensable for antibody‐dependent cellular cytotoxicity by human monocytes. Sci Rep 6:34310, 2016 [00451] 36. Crompton BD, Stewart C, Taylor‐Weiner A, et al: The genomic landscape of pediatric Ewing sarcoma. Cancer Discov 4:1326‐41, 2014 [00452] ***** EQUIVALENTS [00453] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 What is claimed: 1. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, light chain, or a combination thereof, wherein the heavy chain comprises a CDR1 comprising GYSFTNYW (SEQ ID NO: 19), GYSF-(X1X2X3)-W (SEQ ID NO: 13), GYTFTSYY (SEQ ID NO: 47), GFSFSNYG (SEQ ID NO: 51), or GFTF-(X9X10X11)-G (SEQ ID NO: 54), CDR2 comprising IYPGDSDT (SEQ ID NO: 14), INPSGGST (SEQ ID NO: 48), I-( X5)-YDG-(X6X7X8) (SEQ ID NO: 52), or VSHDDINK (SEQ ID NO: 55), and/or CDR3 comprising ARRANPGAFDI (SEQ ID NO: 16), ARPCSAGSCYSTDAFDI (SEQ ID NO: 15), , ARHLRNFGDAFDI (SEQ ID NO: 17), ARDLSSNGDY (SEQ ID NO: 49), ARHLRHFGDAFDV (SEQ ID NO: 50), AR-(X12X13X14)-MDV (SEQ ID NO: 53), or AKGYSGTYGIYFDY (SEQ ID NO: 56); and wherein the light chain comprises a CDR1 comprising SDINVGSYN (SEQ ID NO: 23), KLGNTY (SEQ ID NO: 20), , or ALPKQY (SEQ ID NO: 26), SSDVGGYNY (SEQ ID NO: 55), SGSIATN-(X15) (SEQ ID NO: 58), SLRSYY (SEQ ID NO: 63), or SSNIGSNY (SEQ ID NO: 66), CDR2 comprising YYSDSNK (SEQ ID NO: 24), QD(X4) (SEQ ID NO: 28) , DVS (SEQ ID NO: 56), KDS (SEQ ID NO: 98), EDN (SEQ ID NO: 59), KDD (SEQ ID NO: 61), GKN (SEQ ID NO: 64), RNN (SEQ ID NO: 67), and/or CDR3 comprising MIWHSSAWV (SEQ ID NO: 25), QAWDSSTAV (SEQ ID NO: 22), FSYAGDNRGV (SEQ ID NO: 57), QSYDRTNSVVL (SEQ ID DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 NO: 60), QSYDDGSHYV (SEQ ID NO:62), SSRDSSGNHPYV (SEQ ID NO: 65), or AAWDGSLSGYV (SEQ ID NO: 68). 2. The antibody of claim 1, wherein the antibody is fully human. 3. The antibody of claim 1, wherein the antibody is monospecific or bispecific. 4. The antibody of claim 3, wherein the bispecific target is LINGO1 or IGF1R. 5. The antibody of claim 1, wherein the antibody is a single chain antibody. 6. The antibody of claim 1, wherein the antibody has a binding affinity of at least 3.3x10-9 M. 7. The antibody of claim 1, wherein X1, X2, X3, or X4 is a hydrophilic polar amino acid residue. 8. The antibody of claim 7, wherein the amino acid residue is Threonine (T), Asparagine (N), Lysine (K), Histidine (H), or Serine (S). 9. The antibody of claim 1, wherein X1 is a neutral non-polar amino acid residue. 10. The antibody of claim 9, wherein the amino acid residue is Proline (P) or Tyrosine (Y). 11. The antibody of claim 1, wherein X1X2X3 is STH, PSH, TNY, or SKY. 12. The antibody of claim 1, wherein X4 is S. 13. The antibody of claim 1, wherein X4 is N. 14. The antibody of claim 1, wherein X9 or X10 is a hydrophilic polar amino acid residue. 15. The antibody of claim 15, wherein the amino acid residue is Asparagine (N), Serine (S), Threonine (T), Arginine (R). 16. The antibody of claim 1, wherein X11 is a neutral non-polar amino acid residue. 17. The antibody of claim 16, wherein the amino acid residue is Phenylalanine (F) or Tyrosine (Y). DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 18. The antibody of claim 1, wherein X9X10X11 is RNY, NTF, or SSY. 19. The antibody of claim 1, wherein X5, X6, X7, or X8 is a hydrophilic polar amino acid residue. 20. The antibody of claim 1, wherein the amino acid residue is Asparagine (N), Serine (S), Lysine (K), Histidine (H), Arginine (R), or Glutamate (E). 21. The antibody of claim 1, wherein X5 or X8 is a non-polar amino acid residue. 22. The antibody of claim 21, wherein the amino acid residue is Phenylalanine (F) or Leucine (L). 23. The antibody of claim 1, wherein X5 is Serine (S) or Phenylalanine (F). 24. The antibody of claim 1, wherein X6X7X8 is HKE, SNK, or SRL. 25. The antibody of claim 1, wherein X12 or X13 is a hydrophilic polar amino acid residue. 26. The antibody of claim 25, wherein the amino acid residue is Serine (S) or Arginine (R). 27. The antibody of claim 1, wherein X13 or X14 is a hydrophobic, non-polar amino acid residue. 28. The antibody of claim 27, wherein the amino acid residue is phenylalanine (F) or glycine (G). 29. The antibody of claim 1, wherein X12X13X14 is RGF or SRG. 30. The antibody of claim 1, wherein X15 is a non-polar amino acid residue. 31. The antibody of claim 1, wherein X15 phenylalanine (F), or tyrosine (Y). 32. The antibody according to any one of the preceding claims, wherein the antibody is linked to a therapeutic agent. 33. The antibody of claim 32, wherein the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 34. An antibody composition comprising at least one antibody, wherein the at least one antibody comprises two heavy chains and two light chains, wherein: the heavy chain CDRs are identical to reference germline CDRs found between residues 26 and 33, residues 51 and 58, and residues 97 and 113 according to IMGT numbering of SEQ ID NO: 29, or between residues 97 and 108 according to IMGT numbering of SEQ ID NO: 33, or between residues 97 and 110 according to IMGT numbering of SEQ ID NO: 34, except that at least one of the heavy chain CDRs differs by a single amino acid substitution relative to its reference CDR; and the light chain CDRs are identical to reference germline CDRs found between residues 26 and 31, residues 49 and 51, and residues 88 and 95 according to IMGT numbering of SEQ ID NO:30, or between residues 26 and 34, residues 52 and 58, and residues 97 and 104 according to IMGT numbering of SEQ ID NO: 31, except that at least one of the light chain CDRs differs by a single amino acid substitution relative to its reference CDR, and wherein the antibody composition binds to an epitope that comprises amino residues at positions 23 to 123 of the extracellular domain of CD99 comprising SEQ ID NO: 32. 35. The antibody composition of claim 34, wherein the antibody is fully human. 36. The antibody composition of claim 34, wherein the antibody is a monoclonal antibody. 37. The antibody composition of claim 34, wherein the antibody is an IgG. 38. The antibody composition of claim 34, wherein the antibody is monospecific or bispecific. 39. The antibody composition of claim 34, wherein the antibody has a binding affinity of at least 3.3x10-9 M. 40. The antibody composition of claim 34, wherein the heavy chain CDRs include one or more of the following residue substitutions: 30Pro or 30 or 31Ser; 31Asn or 31or 32Lys; or 32His relative to SEQ ID NO: 29 or SEQ ID NO: 34; or 103Gly relative to SEQ ID NO: 33. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 41. The antibody composition of claim 34, wherein the light chain CDRs include one or more of the following residue substitutions: 26Ala; 28Pro; 29Asn or 29Lys; 30Thr or 30Gln; 51Asn relative to SEQ ID NO: 30; or 57Asn; 100His; 102Ser; or 104Trp relative to SEQ ID NO: 31. 42. A pharmaceutical composition comprising: one or more antibody compositions according to claim 34, and a pharmaceutically acceptable carrier or excipient. 43. The pharmaceutical composition of claim 42, further comprising at least one additional therapeutic agent. 44. The pharmaceutical composition of claim 43, wherein the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine. 45. A cell producing the antibody of any one of claims 1-44. 46. A genetically engineered cell, wherein the genetically engineered cell expresses and/or secretes the antibody of any one of claims 1-44. 47. A method of treating cancer, the method comprising: administering to a subject in need thereof a composition comprising an antibody according to any one of claims 1-41, the composition of claims 42-44, or the cell of claims 45-46. 48. The method of claim 47, wherein the cancer expresses CD99. 49. The method of claim 47, wherein the cancer comprises Ewing Sarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, astrocytoma, glioblastoma, pancreatic endocrine tumor, GI/pulmonary neuroendocrine tumor, prostate cancer, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, lymphoma, or a combination thereof. 50. The method of claim 47, further comprising administering to the subject a chemotherapeutic agent. 51. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 chain comprises an amino acid sequence about 95% identical to SEQ ID NO:3, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:4. 52. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:7, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:8. 53. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:11, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:12. 54. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:71, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:72. 55. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:75, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:76. 56. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD99 comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:79, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:80. 57. A nucleic acid encoding the antibody according to any one of claims 1-41. 58. A nucleic acid encoding the antibody according to any one of claims 51-56. 59. The nucleic acid of claim 58, wherein the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 5 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 6; the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 1 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 2; or the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 9 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 10. 60. The nucleic acid of claim 58, wherein the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 69 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 70; the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 73 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 74; or the nucleic acid encoding the antibody heavy chain comprises SEQ ID NO: 77 and the nucleic acid encoding the antibody light chain comprises SEQ ID NO: 78. 61. A vector comprising the nucleic acid of claims 57 or 58. 62. A cell comprising the vector of claim 61. 63. A kit comprising: the at least one antibody composition of claim 34; a syringe, needle, or applicator for administration of the at least one antibody or fragment thereof to a subject; and instructions for use. 64. A probe comprising the antibody of claim 51, 52, 53, 54, 55, or 56, and wherein the antibody is further coupled to an imaging agent. 65. The antibody composition of claim 40, wherein the heavy chain CDRs include one or more of the following residue substitutions: 30Pro, 32His of SEQ ID NO: 29 relative to SEQ ID NO: 40. 66. The antibody composition of claim 40, wherein the heavy chain CDRs include one or more of the following residue substitutions: 31Asn, 103Gly of SEQ ID NO: 33 relative to SEQ ID NO: 40. 67. The antibody composition of claim 40, wherein the heavy chain CDRs include one or more of the following residue substitutions: 30 or 31Ser; 31or 32Lys of SEQ ID NO: 34 relative to SEQ ID NO: 40. 68. The antibody composition of claim 41, wherein the light chain CDRs include one or more of the following residue substitutions: 29Asn; 30Thr; or 51Asn of SEQ ID NO: 30 relative to SEQ ID NO: 41. DOCKET NO: 5031461-000042-WO1 DATE OF FILING: February 10, 2025 69. The antibody composition of claim 41, wherein the light chain CDRs include one or more of the following residue substitutions: 26Ala; 28Pro; 29Lys; or 30Gln of SEQ ID NO: 110 relative to SEQ ID NO: 41. 70. The antibody composition of claim 41, wherein the light chain CDRs include one or more of the following residue substitutions: 57Asn; 100His; 102Ser; or 104Trp of SEQ ID NO: 31 relative to SEQ ID NO: 42. 71. An isolated anti-CD99 antibody comprising the antibody according to any one of claims 1-41, wherein the anti-CD99 antibody competes with binding to CD99 or binds the same epitope as PILR^ or an anti-PILRα antibody.
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