TUMOR CELL-DERIVED EXOSOMES AND THEIR APPLICATIONS

Abstract

The disclosure features compositions and methods that may be used to detect the presence of tumor cell-derived exosomes in a patient (e.g., a human patient) having cancer. The compositions and methods described herein may also be used to evaluate the patient's prognosis, as well as monitor the likelihood of the patient to benefit from therapy, such as immunotherapy. The disclosure also features antibodies that specifically bind chondroitin sulphate proteoglycan 4 (CSPG4), as well as antigen-antibody complexes containing the same.

Claims

1. A method of detecting the presence of one or more tumor-cell derived exosomes in a patient having melanoma, the method comprising: a. contacting a sample obtained from the patient with antibodies or antigen-binding fragments thereof which specifically bind distinct and spatially distant chondroitin sulphate proteoglycan 4 (CSPG4) epitopes on melanoma exosomes wherein a finding that material bound to the melanoma exosomes, identifies the patient as having melanoma.

2. The method of claim 1, wherein a finding that the material bound to the melanoma exosomes identifies the melanoma as likely to resist detection and/or cell death by the patient's immune system.

3. The method of claim 1, wherein a finding that the material bound to the melanoma exosomes identifies the patient as likely to benefit from treatment with immunotherapy.

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42. The method of claim 1, wherein the patient is a human.

43. An antigen-antibody complex comprising: a. a first antibody, or an antigen-binding fragment thereof, that specifically binds a first epitope on CSPG4; b. a second antibody, or an antigen-binding fragment thereof, that specifically binds a second epitope on CSPG4; and c. a CPSG4 proteoglycan; wherein the first and second epitopes on CSPG4 are different from one another.

44. The antigen-antibody complex of claim 43, wherein the first and second epitopes on CSPG4 do not share any overlapping amino acid residues.

45. The antigen-antibody complex of claim 43, wherein one or both of the first and second antibodies or antigen-binding fragments comprise a detectable label.

46. The antigen-antibody complex of claim 43, wherein the first antibody or antigen-binding fragment comprises a detectable label comprising biotin.

47. The antigen-antibody complex of claim 43, wherein the second antibody or antigen-binding fragment comprises a detectable label comprising a fluorophore.

48. A kit comprising: a. a first antibody, or an antigen-binding fragment thereof, that specifically binds a first epitope on CSPG4; and b. a second antibody, or an antigen-binding fragment thereof, that specifically binds a second epitope on CSPG4; wherein the first and second epitopes on CSPG4 are different from one another, optionally wherein the first and second epitopes on CSPG4 do not share any overlapping amino acid residues.

49. The kit of claim 48, wherein one or both of the first and second antibodies or antigen-binding fragments comprise a detectable label.

50. The kit of claim 48, wherein the first antibody or antigen-binding fragment comprises a detectable label comprising biotin.

51. The kit of claim 49, wherein the kit further comprises a compound that specifically binds the detectable label of the first antibody or antigen-binding fragment.

52. The kit of claim 51, wherein the compound is immobilized to a surface.

53. The kit of claim 52, wherein the surface is a bead.

54. The kit of claim 53, wherein the bead comprises a polysaccharide.

55. The kit of claim 54, wherein the polysaccharide is agarose.

56. The kit of claim 51, wherein the compound comprises avidin or streptavidin.

57. The kit of claim 48, wherein the second antibody or antigen-binding fragment comprises a detectable label comprising a fluorophore.

58. The kit of claim 48, wherein the kit further comprises a filter suitable for ultrafiltration, wherein the filter has a pore size of about 0.2 μm.

59. The kit of claim 48, wherein the kit further comprises one or more size exclusion chromatography columns.

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Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 shows methods for isolation of exosomes from plasma of patients with melanoma by size exclusion chromatography and separation of total exosomes recovered in fractions #3 and 4 into melanoma cell-derived exosomes (MTEX) and nonMTEX by immune capture with biotinylated anti-CSPG4 monoclonal antibodies (mAb). TME tumor microenvironment.

[0040] FIG. 2 shows the specificity of anti-CSPG4 monoclonal antibodies (mAb) clones TP4 and TP6 for detection of this antigen on melanoma cell-derived exosomes (MTEX) and nonMTEX. a,b Nearly all MTEX immunocaptured with clone TP4 mAb and detected with clone TP6 mAb are CSPG4(+); nonMTEX are negative for CSPG4. c,d Nearly all MTEX immunocaptured with clone TP6 and detected with clone TP4 mAb are CSPG4(+); nonMTEX are negative for CSPG4. The CSPG4-specific capture mAbs were used at the protein concentration of 3 μg, while anti-CD63 mAb for capture of nonMTEX was 2 μg. Note that dilu-tions of capture and detection mAbs are critical for the success of capture as well as detection of CSPG4+exosomes. a MTEX: capture antibody (clone VF1-TP41.2)—3 μg, detection antibody (clone TP61.5)—2.5 μg; b nonMTEX: capture antibody (anti-CD63)—2 μg, detection antibody (TP61.5)—0.75 μg; c MTEX: capture antibody (cloneTP61.5)—3 μg, detection anti-body (VF1-TP41.2)—2.5 μg; d nonMTEX: capture antibody (anti-CD63)—2 μg, detection antibody (VF1-TP41.2)—0.75 μg. RFI relative fluorescence index.

[0041] FIG. 3 shows chondroitin sulfate proteoglycan 4 (CSPG4) expression levels on Mel526 cells and on exosomes produced by MEL526 cells. Numbers indicate relative fluorescence index (RFI) values, which are nearly the same for cells and exosomes. a MEL526 cells, b MEL526 exosomes.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Below, we describe:(a) the characteristics of melanoma cell-derived exosomes (MTEX) separated by immunoaffinity capture from exosomes produced by non-malignant cells (non-MTEX); (b) the characteristics of the tumor antigen chondroitin sulfate proteoglycan 4 (CSPG4; Campoli et al., Crit Rev Immunol 24:267-296 (2004); Campoli et al., Adv Cancer Res 109:73-121 (2010)), which we used as a target to separate MTEX from non MTEX; and (c) the evidence supporting the role MTEX play in immune suppression in melanoma.

[0043] Accordingly, we describe the functional properties of the exosomes released from melanoma cells. In particular, we have detailed characteristics of the tumor antigen chondroitin sulfate proteoglycan 4 (CSPG4), which is used as a marker to separate exosomes released by melanoma cells from exosomes released by nonmalignant cells. Our results described herein are useful in view of the role of melanoma cell-derived exosomes in the escape of malignant cells from the host's immune system.

Exosomes Serve as a Communication System Between the Tumor and the Immune System

[0044] Melanoma patients' plasma contains exosomes produced by malignant and non-malignant cells. To analyze the phenotypic and functional characteristics of MTEX, we have developed an immunoaffinity-based method to separate them from nonMTEX. In this method, first the total exosome population is isolated from plasma using size exclusion chromatography (SEC), as described elsewhere (Hong et al., J Extracell Vesicles 5:29289 (2016)). Exosomes recovered in fractions #3,4 are separated using immune capture with the CSPG4-specific mAb into MTEX and nonMTEX, as also previously described (Sharma et al., J Extracell Vesicles 7(1):1435138 (2017)) and as shown in FIG. 1. The immune-captured MTEX are tested by on-bead flow cytometry for the expression of CSPG4 using a CSPG4-specific labeled mAb, which recognizes an epitope distinct and spatially distant from that recognized by the CSPG4-specific mAb used to capture MTEX. The CSPG4 antigen is expressed on MTEX, but is not detected on non-MTEX. Representative results generated by experiments performed with the CSPG4-specific mAb TP41.1 for capture and mAb TP61.1 for detection, which recognize distinct and spatially distant CSPG4 epitopes, are shown in FIG. 2. The data show that MTEX are positive (99%) and nonMTEX are negative for CSPG4. The protein cargo of successfully separated MTEX can now be further examined by on-bead flow cytometry using labeled mAbs that recognize melanoma/associated antigens (MAAs) or other proteins of interest in the MTEX cargo (Sharma et al., Sci Rep 10(10):92 (2019)). The MTEX and nonMTEX fractions can also be used for RNA or DNA extraction or can be co-incubated with various immune or non-immune cell types to determine their abilities to alter functions of recipient cells.

Characteristics of Chondroitin Sulfate Proteoglycan 4

[0045] The rationale for selection of CSPG4 as a target antigen for immune capture of MTEX from melanoma patients' plasma is based on extensive evaluation of its expression on melanoma and normal human tissues. Like CD44, CSPG4 is a member of the CSPG family of cancer-associated proteins; CSPG4 is also known as a high molecular weight-melanoma-associated antigen (HMW-MAA), or neuron-glial antigen 2 (NG2; (Campoli et al., Crit Rev Immunol 24:267-296 (2004); Campoli et al., Adv Cancer Res 109:73-121 (2010))). The CSPG family members are key bioactive molecules that play a major role in tumor growth, migration, and neo-angiogenesis.

Chondroitin Sulfate Proteoglycan 4 is Highly Expressed on Melanoma Cells

[0046] CSPG4 is highly expressed on melanoma cells in about 80% of primary and metastatic tumors with limited inter- and intralesional heterogeneity (Campoli et al., Crit Rev Immunol 24:267-296 (2004); Campoli et al., Adv Cancer Res 109:73-121 (2010)). It is expressed not only on differentiated melanoma cells, but also on malignant melanoma initiating cells (MMICs). The latter are defined as cells that can form spheres in vitro and are highly tumorigenic in immunodeficient mice. These cells express high levels of aldehyde dehydrogenase and are stained by an ABCB5-specific mAb RK1 (data not shown). In addition, as shown in FIG. 3, exosomes isolated from the spent medium of cultured melanoma cells by sequential differential centrifugation, filtration through a 2μ filter, and size exclusion chromatography are stained by CSPG4-specific mAbs with high intensity. As depicted in FIG. 3, exosomes were captured with biotinylated anti-63 mAb from supernatants of MEL526 cells, and on bead-cytometry was used for detection of CSPG4 on exosomes as described elsewhere (Sharma et al., J Extracell Vesicles 7(1):1435138 (2017)). Mel526 cells expressed high levels of surface CSPG4, and the exosomes these cells produce also carried high levels of CSPG4.

[0047] Data about the expression of CSPG4 in normal tissues are conflicting. In our own experience (Campoli et al., Crit Rev Immunol 24:267-296 (2004); Campoli et al., Adv Cancer Res 109:73-121 (2010); Wang et al., Cancer Res 71:7410-7422 (2011)), immuno-histochemical staining with mAbs that recognize distinct epitopes of CSPG4 has not detected expression of this antigen in any normal tissue with the exception of activated pericytes in the TME (Schlingemann et al., Am J Pathol 136:1393-1405 (1990); Maciag et al., Cancer Res 68:8066-8075 (2008)). Similar results have been reported by Morgan and his collaborators (Beard et al., J Immunother Cancer 2:25 (2014); Beard et al., Clin Cancer Res 19(18):4941-4950 (2013)) using different techniques. This conclusion has been corroborated by several lines of evidence. First, by analyzing 94 normal tissues from different organs with a reverse protein assay, we could detect CSPG4 expression above the threshold level in only two out of the four small-bowel samples tested. Second, no toxicity was detected: (a) in mice injected with large amounts of CSPG4-specific mAb cross-reacting with the mouse CSPG4 homologue (Wang et al., J Natl Cancer Inst 102:1496-1512 (2010)) and (b) in patients and dogs with melanoma as well as in rats with a chemically induced chondrosarcoma (Mittelman et al., Proc Natl Acad Sci USA 89:466-470 (1992); Riccardo et al., Clin Cancer Res 20:3753-3762 (2014); Léger et al., Int J Cancer 58(5):700-705 (1994)) who developed CSPG4-specific antibodies following immunizations with CSPG4 mimics. Lastly, CSPG4-specific CAR+T cells did not lyse various types of normal cells that are not stained by CSPG4-specific mAbs (Geldres et al., Clin Cancer Res 20:962-971 (2014)).

[0048] In contrast to our results summarized above, the data reported in the Protein Atlas, which have been obtained utilizing commercially available anti-CSPG4 antibodies, indicated that CSPG4 has a broad distribution in normal tissues. This conflicting evidence is likely caused by the lack of specificity of some of the commercial antibodies used to generate the data presented in the Protein Atlas. For instance, the rabbit antiserum provided by Sigma does not appear to be specific for CSPG4, since it recognizes a molecule with a molecular weight different from that of CSPG4 in Western blotting. Furthermore, the same antibody reacts with cells in which CSPG4 has been knocked out by CRISPR. The conflict that exists between our data and recommendations published in the Protein Atlas has led to confusion among investigators using commercial anti-CSPG4 mAbs for immune capture. We emphasize the specificity of our anti-CSPG4 mAbs for epitopes overexpressed on melanoma (or other tumor) cells and the lack of their reactivity with normal human tissues. Such specificity is useful for immune capture of CSPG4+ cells or exosomes. Some of the commercially available mAbs targeting CSPG4 fail to meet similar standards for tumor cell specificity and thus cannot be reliably used for immune capture of MTEX.

[0049] In sum, the tumor antigen CSPG4 is highly expressed on melanoma cells and is thus used as a marker to separate melanoma cell-derived exosomes (MTEX) from exosomes released form nonmalignant cells (nonMTEX).

[0050] A comparison of the phenotype and functional properties of MTEX and nonMTEX has shown that MTEX carry an abundance of immunosuppressive proteins and inhibit numerous functions of human primary T cells and natural killer cells. As a result, MTEX may promote tumor immune escape and tumor progression.

[0051] NonMTEX that are enriched in co-stimulatory proteins might stimulate immune cell activity.

[0052] Furthermore, we have demonstrated the separation of MTEX from nonMTEX fractions of exosomes in plasma of melanoma patients. The ability to perform this separation by immunoaffinity capture permits measuring the ratios of MTEX/nonMTEX in plasma of patients with metastatic melanoma. We have found that this ratio may vary from 20 to 60% (Sharma et al., Sci Rep 10(10):92 (2019)). In addition, we have been able to compare the phenotype and functional properties of MTEX and nonMTEX (Sharma et al., Sci Rep 10(10):92 (2019)). This comparison has shown that MTEX carry an abundance of immunosuppressive proteins and inhibit numerous functions of human primary T cells and natural killer (NK) cells ex vivo, as also described elsewhere (Wieckowski et al., J Immunol 183(6):3720-3730 (2009)). As a result, MTEX may promote tumor immune escape and tumor progression. By contrast, nonMTEX that are enriched in co-stimulatory proteins might stimulate immune cell activity (Sharma et al., Sci Rep 10(10):92 (2019); Sharma et al., J Extracell Vesicles 7(1):1435138 (2017)).

[0053] It is noteworthy that CSPG4 is also expressed on cancer cells in glioma, head and neck cancer, mesothelioma, triple-negative breast cancer, ovarian cancer, and malignancies of orthopedic interest (Campoli et al., Crit Rev Immunol 24:267-296 (2004); Campoli et al., Adv Cancer Res 109:73-121 (2010)). Therefore, the methodology we have developed for isolation of MTEX is applicable to other human malignancies.

[0054] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0055] The invention is also characterized by the following enumerated embodiments:

[0056] 1. A method of detecting the presence of one or more tumor-cell derived exosomes in a patient having cancer, the method comprising: [0057] a. contacting a sample obtained from the patient with an antibody, an antigen-binding fragment thereof, or a ligand that specifically binds chondroitin sulphate proteoglycan 4 (CSPG4); [0058] b. isolating the antibody, antigen-binding fragment, or ligand from the sample; and [0059] c. analyzing material bound to the antibody, antigen-binding fragment, or ligand for the presence of CSPG4;

[0060] wherein a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the patient as having one or more tumor-cell derived exosomes.

[0061] 2. The method of embodiment 1, wherein a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the cancer as likely to resist detection and/or cell death by the patient's immune system.

[0062] 3. The method of embodiment 1 or 2, wherein a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the patient as likely to benefit from treatment with immunotherapy.

[0063] 4. A method of determining whether a cancer in a patient suffering therefrom is likely to resist detection and/or cell death by the patient's immune system, the method comprising: [0064] a. contacting a sample obtained from the patient with an antibody, an antigen-binding fragment thereof, or a ligand that specifically binds CSPG4; [0065] b. isolating the antibody, antigen-binding fragment, or ligand from the sample; and [0066] c. analyzing material bound to the antibody, antigen-binding fragment, or ligand for the presence of CSPG4;

[0067] wherein a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the cancer as likely to resist detection and/or cell death by the patient's immune system.

[0068] 5. The method of embodiment 4, wherein a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the patient as having one or more tumor-cell derived exosomes.

[0069] 6. The method of embodiment 4 or 5, wherein a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the patient as likely to benefit from treatment with immunotherapy.

[0070] 7. A method of determining whether a patient having cancer is likely to benefit from treatment with immunotherapy, the method comprising: [0071] a. contacting a sample obtained from the patient with an antibody, an antigen-binding fragment thereof, or a ligand that specifically binds CSPG4; [0072] b. isolating the antibody, antigen-binding fragment, or ligand from the sample; and [0073] c. analyzing material bound to the antibody, antigen-binding fragment, or ligand for the presence of CSPG4;

[0074] wherein a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the patient as likely to benefit from treatment with immunotherapy.

[0075] 8. The method of embodiment 7, wherein a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the patient as having one or more tumor-cell derived exosomes.

[0076] 9. The method of embodiment 7 or 8, wherein a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the cancer as likely to resist detection and/or cell death by the patient's immune system.

[0077] 10. The method of any one of embodiments 1-9, wherein, prior to contacting the sample with the antibody, antigen-binding fragment, or ligand, the sample is subjected to ultrafiltration through a filter having a pore size of about 0.2 μm.

[0078] 11. The method of any one of embodiments 1-10, wherein, prior to contacting the sample with the antibody, antigen-binding fragment, or ligand, the sample is subjected to differential centrifugation.

[0079] 12. The method of any one of embodiments 1-11, wherein, prior to contacting the sample with the antibody, antigen-binding fragment, or ligand, the sample is subjected to size exclusion chromatography.

[0080] 13. The method of any one of embodiments 1-12, wherein, prior to contacting the sample with the antibody, antigen-binding fragment, or ligand the sample is prepared by: [0081] a. subjecting the sample to differential centrifugation, subsequently [0082] b. subjecting the sample to ultrafiltration through a filter having a pore size of about 0.2 μm, and subsequently [0083] c. subjecting the sample to size exclusion chromatography.

[0084] 14. A method of purifying one or more tumor-cell derived exosomes in a sample obtained from a patient having cancer, the method comprising: [0085] a. subjecting the sample to differential centrifugation, ultrafiltration, and/or size exclusion chromatography; [0086] b. contacting the sample with an antibody, an antigen-binding fragment thereof, or a ligand that specifically binds CSPG4; and [0087] c. isolating the antibody, antigen-binding fragment, or ligand from the sample;

[0088] wherein the method optionally comprises analyzing material bound to the antibody, antigen-binding fragment, or ligand for the presence of CSPG4.

[0089] 15. The method of any one of embodiments 1-14, wherein the antibody, antigen-binding fragment, or ligand comprises a detectable label, and wherein the antibody, antigen-binding fragment, or ligand is isolated from the sample by: [0090] a. contacting the antibody, antigen-binding fragment, or ligand with a compound that specifically binds the detectable label, and subsequently [0091] b. separating the compound from the sample.

[0092] 16. The method of embodiment 15, wherein the compound is immobilized to a surface.

[0093] 17. The method of embodiment 16, wherein the surface is a bead.

[0094] 18. The method of embodiment 17, wherein the bead comprises a polysaccharide.

[0095] 19. The method of embodiment 18, wherein the polysaccharide is agarose.

[0096] 20. The method of any one of embodiments 15-19, wherein the detectable label comprises biotin.

[0097] 21. The method of any one of embodiments 15-20, wherein the compound comprises avidin or streptavidin.

[0098] 22. The method of any one of embodiments 1-21, wherein the cancer is melanoma, glioma, head and neck cancer, mesothelioma, breast cancer, or ovarian cancer, optionally wherein the breast cancer is triple negative breast cancer.

[0099] 23. The method of embodiment 22, wherein the cancer is melanoma.

[0100] 24. The method of any one of embodiments 1-3, 5, 6, and 8-23, wherein the one or more tumor cell-derived exosomes are melanoma cell-derived exosomes.

[0101] 25. The method of any one of embodiments 1-3, 5, 6, and 8-24, wherein the one or more tumor cell-derived exosomes have a size of from about 30 to about 150 nm.

[0102] 26. The method of any one of embodiments 1-3, 5, 6, and 8-25, wherein the one or more tumor cell-derived exosomes comprise one or more enzymes, growth factors, oncogenes and signaling immunoregulatory proteins.

[0103] 27. The method of any one of embodiments 1-3, 5, 6, and 8-26, wherein the one or more tumor cell-derived exosomes comprise one or more immunosuppressive proteins.

[0104] 28. The method of embodiment 27, wherein the one or more immunosuppressive proteins comprise Fas ligand (FasL), transforming growth factor-beta (TGF-beta), TNF superfamily member 10 (TRAIL), and/or programmed death-ligand 1 (PD-L1).

[0105] 29. The method of any one of embodiments 1-28, wherein the antibody, antigen-binding fragment, or ligand specifically binds a first epitope on CSPG4, and wherein the material bound to the antibody, antigen-binding fragment, or ligand is analyzed for the presence of CSPG4 by contacting the material with an antibody, or an antigen-binding fragment thereof, that specifically binds a second epitope on CSPG4, wherein the first and second epitopes on CSPG4 are different from one another.

[0106] 30. The method of embodiment 29, wherein the first and second epitopes on CSPG4 do not share any overlapping amino acid residues.

[0107] 31. The method of embodiment 29 or 30, wherein the antibody or antigen-binding fragment that specifically binds the second epitope on CSPG4 comprises a detectable label.

[0108] 32. The method of embodiment 31, wherein the detectable label on the antibody or antigen-binding fragment that specifically binds the second epitope on CSPG4 comprises a fluorophore.

[0109] 33. The method of embodiment 32, wherein the presence of CSPG4 is signaled by a finding of fluorescence at an emission wavelength characteristic of the fluorophore.

[0110] 34. The method of any one of embodiments 1-33, wherein the sample comprises blood plasma.

[0111] 35. A method of determining whether a patient having cancer and that has been administered one or more therapeutic agents is benefiting from treatment with the one or more therapeutic agents, optionally wherein the one or more therapeutic agents comprise an immunotherapy, the method comprising: [0112] a. contacting a sample obtained from the patient with an antibody, an antigen-binding fragment thereof, or a ligand that specifically binds CSPG4; [0113] b. isolating the antibody, antigen-binding fragment, or ligand from the sample; and [0114] c. analyzing material bound to the antibody, antigen-binding fragment, or ligand for the presence of CSPG4;

[0115] wherein a finding that the quantity of CSPG4 in the sample has decreased relative to a previous measurement of CSPG4 in the patient is taken as an indication that the patient is benefiting from the treatment.

[0116] 36. A method of treating a cancer in a patient in need thereof, the method comprising administering to the patient an immunotherapy, wherein the patient has been selected for treatment with an immunotherapy by the method of any one of embodiments 3 and 6-34.

[0117] 37. The method of any one of embodiments 3 and 6-36, wherein the immunotherapy comprises an antibody, or an antigen-binding fragment thereof, that specifically binds an immune checkpoint protein.

[0118] 38. The method of embodiment 37, wherein the immune checkpoint protein is CTLA-4.

[0119] 39. The method of embodiment 38, wherein the immunotherapy comprises ipilimumab.

[0120] 40. The method of embodiment 37, wherein the immune checkpoint protein is PD-1.

[0121] 41. The method of embodiment 40, wherein the immunotherapy comprises pembrolizumab and/or nivolumab.

[0122] 42. The method of any one of embodiments 1-41, wherein the patient is a human.

[0123] 43. An antigen-antibody complex comprising: [0124] a. a first antibody, or an antigen-binding fragment thereof, that specifically binds a first epitope on CSPG4; [0125] b. a second antibody, or an antigen-binding fragment thereof, that specifically binds a second epitope on CSPG4; and [0126] c. a CPSG4 proteoglycan;

[0127] wherein the first and second epitopes on CSPG4 are different from one another.

[0128] 44. The antigen-antibody complex of embodiment 43, wherein the first and second epitopes on CSPG4 do not share any overlapping amino acid residues.

[0129] 45. The antigen-antibody complex of embodiment 43 or 44, wherein one or both of the first and second antibodies or antigen-binding fragments comprise a detectable label.

[0130] 46. The antigen-antibody complex of any one of embodiments 43-45, wherein the first antibody or antigen-binding fragment comprises a detectable label comprising biotin.

[0131] 47. The antigen-antibody complex of any one of embodiments 43-46, wherein the second antibody or antigen-binding fragment comprises a detectable label comprising a fluorophore.

[0132] 48. A kit comprising: [0133] a. a first antibody, or an antigen-binding fragment thereof, that specifically binds a first epitope on CSPG4; and [0134] b. a second antibody, or an antigen-binding fragment thereof, that specifically binds a second epitope on CSPG4;

[0135] wherein the first and second epitopes on CSPG4 are different from one another, optionally wherein the first and second epitopes on CSPG4 do not share any overlapping amino acid residues.

[0136] 49. The kit of embodiment 48, wherein one or both of the first and second antibodies or antigen-binding fragments comprise a detectable label.

[0137] 50. The kit of embodiment 48 or 49, wherein the first antibody or antigen-binding fragment comprises a detectable label comprising biotin.

[0138] 51. The kit of embodiment 49 or 50, wherein the kit further comprises a compound that specifically binds the detectable label of the first antibody or antigen-binding fragment.

[0139] 52. The kit of embodiment 51, wherein the compound is immobilized to a surface.

[0140] 53. The kit of embodiment 52, wherein the surface is a bead.

[0141] 54. The kit of embodiment 53, wherein the bead comprises a polysaccharide.

[0142] 55. The kit of embodiment 54, wherein the polysaccharide is agarose.

[0143] 56. The kit of any one of embodiments 51-55, wherein the compound comprises avidin or streptavidin.

[0144] 57. The kit of any one of embodiments 48-56, wherein the second antibody or antigen-binding fragment comprises a detectable label comprising a fluorophore.

[0145] 58. The kit of any one of embodiments 48-57, wherein the kit further comprises a filter suitable for ultrafiltration, wherein the filter has a pore size of about 0.2 μm.

[0146] 59. The kit of any one of embodiments 48-58, wherein the kit further comprises one or more size exclusion chromatography columns.

[0147] 60. The kit of any one of embodiments 48-59, wherein the kit further comprises a package insert instructing a user of the kit to perform the method of any one of embodiments 1-42.

[0148] 61. Use of an antibody, an antigen-binding fragment thereof, or a ligand that specifically binds CSPG4 in the manufacture of a kit for performing the method of any one of embodiments 1-42.