Oncologic multi-epitope general antibodies

Abstract

Methods of treating cancer in a subject comprising receiving plasmablasts and / or plasma cells from a tumor or malignant effusion, culturing the received cells in culture media, isolating polyclonal antibodies and administering the antibodies to the subject, thereby treating cancer are provided. Compositions comprising polyclonal antibodies from B cells, plasmablasts and / or plasma cells derived from a tumor or malignant effusion are also provided as are methods of enriching tumor derived B cells, plasmablasts and / or plasma cells in culture.

Description

ONCOLOGIC MULTI-EPITOPE GENERAL ANTIBODIES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Israeli Patent Application No.318455 filed January 16, 2025, the contents of which are all incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (MOR-P-031 -PCT. xml; Size: 4,168 bytes; and Date of Creation: November 16, 2025) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of cancer diagnostics.BACKGROUND OF THE INVENTION

[0004] Antibody therapies revolutionized clinical oncology. Monoclonal antibodies can be developed to target tumor associated antigens and therefore offer a flexible targeting of known antigens. However, these therapies are restricted to application in patients with high expression of the targeted antigen. In addition, tumor heterogeneity limits efficacy. Consequently, targeting a specific tumor associated antigen is commonly associated with tumor escape due to phenotypic plasticity of cancer cells and selective pressure resulting from treatment with the therapeutic monoclonal antibody.

[0005] T cells and B cells express receptors or secrete antibodies that recognize specific target antigens. Each cell may express a different receptor or antibody. Monoclonal treatments leverage only one specific cell or antibody to target a known antigen. Thus, these treatments are susceptible to escape and may trigger selection mechanisms leading to treatment resistance.

[0006] Therefore, polyclonal therapies were developed in the field of oncology, rheumatology and immune deficiencies. Specifically, Tumor Infiltrating Lymphocytes (TILs) were recently clinically approved for metastatic melanoma. The methodology of these autologous treatments relies on expanding tumor infiltrating T cells ex -vivo and reinfusing them back into the patients. Consequently, TIL therapy is based on polyclonal T cells with unknown specific anti-tumor antigens binding. TIL therapy is limited by manufacturing constraints associated with autologous cell therapy treatments and it requires crucial patient selection to ensure T cell extraction. Beyond T cell therapies, there are no polyclonal therapies for the treatment of malignancies to date. Conversely, Intravenous Immunoglobulin (IVIg) therapies are utilized for the treatment of immune deficiencies and autoimmune diseases. IVIg is based on pooled, highly polyclonal antibodies extracted from healthy donors with the aim of normalizing a compromised immune system. However, to date, IVIg is not employed for the treatment of cancer.

[0007] Recently, B cells and antibody producing cells were shown to infiltrate tumors, and to be associated with higher survival and response to immune checkpoint therapy. The B cell lineage includes B cell subsets, plasma blasts and plasma cells. B cells are the original cells of interest expressing antibodies (in a transmembrane form). Upon differentiation to plasma blasts or plasma cells, the cells secrete large amounts of antibodies. Differentiation towards antibody secreting cells is common in tumors bearing Tertiary Lymphoid Structures (TLS). TLSs are organized sites of lymphatic interactions enabling an in-situ immune response. In mechanisms that were found to be similar to mechanisms occurring in primary and secondary lymphatic tissues such as lymph nodes or spleen, B cells in TLS underwent antigen induced activation, proliferation and differentiation.

[0008] To conclude, tumor escape to monoclonal therapies is common. Polyclonal therapies are either based on cell therapeutics, which are highly troublesome to manufacture, or IVIg, which is not employed to treat malignancies. A new polyclonal therapy for treating malignancies that makes use of polyclonal antibodies and not cells is therefore greatly needed.SUMMARY OF THE INVENTION

[0009] The present invention provides methods of treating cancer in a subject comprising receiving plasmablasts and / or plasma cells from a tumor or malignant effusion, culturing thereceived cells in culture media, isolating polyclonal antibodies and administering the antibodies to the subject, thereby treating cancer. Compositions comprising polyclonal antibodies from B cells, plasmablasts and / or plasma cells derived from a tumor or malignant effusion are also provided as are methods of enriching tumor derived B cells, plasmablasts and / or plasma cells in culture.

[0010] According to a first aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising:a. receiving plasmablasts and / or plasma cells from a tumor or malignant effusion from a subject suffering from cancer;b. culturing the received plasmablasts or plasma cells in culture media;c. isolating a composition of polyclonal antibodies from the culture media;andd. administering the composition of polyclonal antibodies to the subject in need thereof;thereby treating cancer in a subject in need of treatment.[Oil] According to some embodiments, the method comprises receiving plasmablasts and / or plasma cells from a malignant effusion from the subject suffering from cancer.

[0012] According to some embodiments, the cancer in the subject in need of treatment is the same type of cancer as the tumor or the cancer that produced the malignant effusion.

[0013] According to some embodiments, the cancer in the subj ect in need of treatment is the same type of cancer as the cancer that produced the malignant effusion.

[0014] According to some embodiments, the cancer in the subject in need of treatment is a different type of cancer as the tumor or the cancer that produced the malignant effusion.

[0015] According to some embodiments, the cancer in the subject in need of treatment is a different type of cancer as the cancer that produced the malignant effusion.

[0016] According to some embodiments, the cancer is selected from renal cancer, liver cancer, pancreatic cancer, lung cancer, gastric cancer, ovarian cancer, skin cancer, head and neck cancer, uterine cancer and cervical cancer.

[0017] According to some embodiments, the cancer is selected from renal cancer, liver cancer, pancreatic cancer, lung cancer, gastric / gastrointestinal cancer, ovarian cancer, breast cancer, skin cancer, head and neck cancer, uterine cancer, brain cancer, bladder cancer and cervical cancer.

[0018] According to some embodiments, the renal cancer is renal cell carcinoma (RCC), or the liver cancer is hepatocellular carcinoma (HCC).

[0019] According to some embodiments, the receiving comprises receiving a tumor sample or malignant effusion sample, isolating CD 19 positive cells from the sample and culturing the isolated CD 19 cells in media to enrich for plasmablasts and plasma cells.

[0020] According to some embodiments, the receiving comprises receiving a malignant effusion sample, isolating CD19 positive cells from the sample and culturing the isolated CD 19 cells in media to enrich for plasmablasts and plasma cells.

[0021] According to some embodiments, the receiving comprises receiving a tumor sample or malignant effusion sample, and isolating cells positive for syndecan 1 (CD138 / SDC1), B-cell maturation antigen (BCMA / TBFRSF17), CD38 or a combination thereof from the sample, thereby isolated plasmablasts and / or plasma cells.

[0022] According to some embodiments, the receiving comprises receiving a malignant effusion sample, and isolating cells positive for syndecan 1 (CD138 / SDC1), B-cell maturation antigen (BCMA / TBFRSF17), CD38 or a combination thereof from the sample, thereby isolated plasmablasts and / or plasma cells.

[0023] According to some embodiments, the receiving comprises receiving a tumor sample or malignant effusion sample, isolating immune cells from the sample, culturing the isolated immune cells in B cell media for a time sufficient to expand a CD 19+ B cell population, isolating CD 19 positive cells from the culture and further culturing the isolated CD 19 positive cells to produce plasmablasts and plasma cells.

[0024] According to some embodiments, the receiving comprises receiving a malignant effusion sample, isolating immune cells from the sample, culturing the isolated immune cells in B cell media for a time sufficient to expand a CD19+ B cell population, isolating CD19 positive cells from the culture and further culturing the isolated CD 19 positive cells to produce plasmablasts and plasma cells.

[0025] According to some embodiments, the method does not comprise a step of enriching for 4-1 BBL positive cells.

[0026] According to some embodiments, the method does not comprise isolating antibodies from immortalized cells.

[0027] According to some embodiments, the method does not comprise a step of immortalizing cells.

[0028] According to some embodiments, the malignant effusion is selected from an ascites and a pleural effusion.

[0029] According to some embodiments, the media is B cell culture media.

[0030] According to some embodiments, the enriching comprises culturing for an amount of time sufficient to produce a culture comprising at least 30% plasmablasts and plasma cells.

[0031] According to some embodiments, the method comprises performing steps a-c with plasmablasts and / or plasma cells from a malignant effusion from a plurality of subjects suffering from cancer, wherein the plurality comprises a first subject suffering from cancer and a second subject suffering from cancer, wherein the first and second subject are different subjects, and wherein the administering comprising administering polyclonal antibody isolated from the received plasmablasts and / or plasma cells from the plurality of subjects.

[0032] According to some embodiments, the plurality of subjects all suffer from the same type of cancer as the subject in need of treatment.

[0033] According to some embodiments, the method further comprises genetically engineering the B cells, plasmablasts or plasma cells during the culturing.

[0034] According to some embodiments, the genetic engineering comprises linking an exogenous protein to an endogenous heavy chain or light chain constant region or fragment thereof.

[0035] According to some embodiments, the genetic engineering comprises RNA-guided nuclease engineering, optionally wherein the RNA-guided nuclease engineering is CRISPR-Cas engineering.

[0036] According to some embodiments, the genetic engineering comprises linking a T cell engager to an endogenous heavy chain or light chain constant region or fragment thereof.

[0037] In some embodiments, the exogenous protein is selected from a T cell engager, a cytokine or chemokine and an ant-immune protein antibody or antigen binding fragment thereof.

[0038] According to some embodiments, the T cell engager is an anti-CD3 single chain antibody or anti-CD3 single domain antibody.

[0039] According to some embodiments, the T cell engager is an anti-CD3 single chain antibody and comprises an amino acid sequence selected from SEQ ID NO: 1-3.

[0040] According to some embodiments, the cytokine or chemokine is selected from interleukin 15 (IL- 15), IL-7, IL-2, IL-21, IL- 12, IL-4, C-X-C motif chemokine 12 (CXCL12), and CXCL13.

[0041] According to some embodiments, the anti-immune protein antibody is an antibody against an immune protein selected from CD28, CD40, Programmed death-ligand 1 (PD-Ll), Cytotoxic T-lymphocyte associated protein 4 (CTLA4), C-X-C chemokine receptor type 4 (CXCR4), and CXCR5.

[0042] According to some embodiments, the genetic engineering comprises introducing at least one first mutation into an endogenous heavy chain constant region, introducing a sequence encoding an anti-CD3 antibody’s light chain, introducing a sequence encoding the anti-CD3 antibody’s heavy chain and wherein the anti-CD3 antibody’s heavy chain comprises at least one second mutation and wherein the first and second mutations induce heterodimerization between the endogenous heavy chain constant region and the anti-CD3 antibody’s heavy chain and inhibit homodimerization.

[0043] According to some embodiments, the genetic engineering comprises introducing at least one first mutation into an endogenous heavy chain constant region, introducing a sequence encoding an anti -immune protein antibody’s light chain, introducing a sequence encoding the anti-immune protein antibody’s heavy chain and wherein the anti -immune protein antibody’s heavy chain comprises at least one second mutation and wherein the first and second mutations induce heterodimerization between the endogenous heavy chain constant region and the anti-immune protein antibody’s heavy chain and inhibit homodimerization.

[0044] According to some embodiments, the first and second mutations are selected from Table 1.

[0045] According to some embodiments, the genetic engineering comprises introducing a sequence encoding an anti-CD3 antibody’s light chain and introducing a sequence encoding the anti-CD3 antibody’s heavy chain, whereina. a CL domain of the anti-CD3 antibody ’ s light chain has been replaced with a CHI domain and a CHI domain of the anti-CD3 antibody’s heavy chain has been replaced with a CL domain; orb. a CL domain of an endogenous light chain has been replaced with a CHI domain and a CHI domain of an endogenous heavy chain has been replaced with a CL domain.

[0046] According to some embodiments, the genetic engineering comprises introducing a sequence encoding an anti -immune protein antibody’s light chain and introducing a sequence encoding the anti-immune protein antibody’s heavy chain, whereina. a CL domain of the anti -immune protein antibody’s light chain has been replaced with a CHI domain and a CHI domain of the anti-immune protein antibody’s heavy chain has been replaced with a CL domain; orb. a CL domain of an endogenous light chain has been replaced with a CHI domain and a CHI domain of an endogenous heavy chain has been replaced with a CL domain.

[0047] According to some embodiments, the method further comprises isolating the polyclonal antibodies from the culture media.

[0048] According to some embodiments, the composition is a pharmaceutical composition comprising the polyclonal antibodies and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0049] According to some embodiments, the method comprises loading the isolated polyclonal antibodies onto a population of T cells expressing an anti-Fc receptor chimeric antigen receptor (CAR) to produce a population of polyclonal antibody loaded T cells and wherein the administering is administering the population of polyclonal antibody loaded T cells.

[0050] According to some embodiments, the method comprises conjugating a drug to the isolated polyclonal antibodies to produce a polyclonal antibody-drug conjugate (ADC) composition and wherein the administering is administering the ADC composition.

[0051] According to some embodiments, the pharmaceutical composition consists of the polyclonal antibodies and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0052] According to another aspect, there is provided a pharmaceutical composition comprising polyclonal antibodies harvested from B cells, plasmablasts and / or plasma cells derived from a tumor or malignant effusion in culture and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0053] According to some embodiments, the pharmaceutical composition is for use in treating cancer.

[0054] According to another aspect, there is provided a method of enriching tumor derived B cells, plasmablasts and / or plasma cells in culture, the method comprising receiving a mixed population of immune cells from a tumor, culturing the mixed population in culture media for a time sufficient to enrich B cells, plasmablasts and / or plasma cells in the culture, wherein the culture is selected from:a. Immunocult Human B cell expansion media;b. Immunocult Human B cell expansion kit supplemented with interleukin 21 (IL-21);c. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, RP105, and IL-7;d. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, RP105, and IL-4;e. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, RP105, IL-4, and IL-21;f. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, megaCD40L, CpG ODN2006, IL-10, IL-2, IL-15, and IL-6;g. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, megaCD40L, IL-4, IL-21, and B-cell activating factor (BAFF / TNFSF13B);h. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, megaCD40L, and 1 IL-4; andi. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, HEPES, R848, IL-2, IL- 10, and IL-21.

[0055] According to another aspect, there is provided a method of enriching tumor derived B cells, plasmablasts and / or plasma cells in culture, the method comprising receiving a mixed population of primary immune cells from a tumor, culturing the mixed population in culture media for a time sufficient to enrich B cells, plasmablasts and / or plasma cells in the culture, wherein the culture is selected from:a. Immunocult Human B cell expansion media;b. Immunocult Human B cell expansion kit supplemented with interleukin 21 (IL-21);c. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, RP105, and IL-7;d. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, RP105, and IL-4;e. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, RP105, IL-4, and IL-21;f. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, megaCD40L, CpG ODN2006, IL-10, IL-2, IL-15, and IL-6;g. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, megaCD40L, IL-4, IL-21, and B-cell activating factor (BAFF / TNFSF13B);h. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, megaCD40L, and 1 IL-4; and

[0056] RPMI 1640 supplemented with FBS, beta-mercaptoethanol, HEPES, R848, IL-2, IL-10, and IL-21.

[0057] According to some embodiments, the culturing comprises culturing for time sufficient for expansion of a CD 19 positive B cell population, isolating CD 19 positive cells from the culture and further culturing the isolated CD 19 positive cells to produce plasmablasts and plasma cells and wherein the method is a method of enriching tumor derived plasmablasts and / or plasma cells.

[0058] According to some embodiments, the mixed population is depleted of CD3 positive cells.

[0059] According to some embodiments, the method does not comprise a step of enriching for 4-1 BBL positive cells.

[0060] According to some embodiments, the method does not comprise a step of isolating antibodies from immortalized cells. According to some embodiments, the method does not comprise a step of immortalizing cells.

[0061] According to another aspect, there is provided a population of tumor derived B cells, plasmablasts and / or plasma cells produced by a method of the invention.

[0062] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1: Overview OMG Antibody therapy process. A schematic of an embodiment of the proposed OMG antibody pipeline. The original patient has a tumor (orange cells) infiltrated with B lineage cells (colorful cells). The B cells and antibody secreting cells are collected and cultured ex -vivo. From the polyclonal culture, secreted polyclonal antibodies are collected and purified. The antibodies are then utilized as a therapy for multiple patients either with the same tumor type as the original patient (orange) or for other tumor types with possible cross-response (yellow and red).

[0064] Figures 2A-2E: Characterization of B cells from peripheral blood. (2A) Recovery rates of B cells from PBMCs of distinct individuals. The rates represent the number of effective cells after negative selection for B lineage cells, compared to the expected absolute B cell number in the original PBMC sample. (2B) Frequency of B cells before (pre) and after (post) negative enrichment of PBMC cells from healthy donor samples.(2C) Viability of enriched B lineage culture over time from 3 healthy donors, as assessed by trypan blue. Each color represents a different sample from distinct individuals. (2D) Expected expansion of B lineage cells in culture as calculated for a 100ml original blood sample and cultured over time as described in 2C. Indicated by the colors are Low (orange), Middle (blue) or High (red) expansion values as calculated by values of B cell frequency in blood sample, recovery rate and expansion rate, where values of expansion were utilized from the 3 donors in 2A-2C. (2E) Spectral cytometry characterization of B lineage cells in culture at day 0, 8 and 14. From left to right: migratory B cells (CXCR4+ CXCR5+ from CD19+), antigen presenting B cells (CXCR4+ CD86+ from CD19+), Plasmablasts (CD138+ CD38+) and early antibody secreting cells (CD27+ CD38+).

[0065] Figures 3A-3G: Characterization of tumor infiltrating antibody expressing cells extraction, culture and antibody production. (3A) Experimental scheme of solid tumor processing. Tumor samples are fragmented and cultured in B lineage or T cell supporting growth media. Tumor fragments are then removed and lymphocyte culture proceeds in culture conditions. (3B) White field microscopy (left) and cartoon representation of the same field (right) of an example tumor fragmented in culture demonstrating lymphocyte extravagation from the tumor fragment (represented in pink). TILs are either cultured in IL-2, supporting T cell growth, or B cell expansion media, supporting B lineage cells. In the cartoon representation, lymphocytes are represented in green or blue, dead cells or debris in red and culture media supplements in orange. (3C) Comparison of the frequency of CD4, CD8 and CD 19 cells in samples as described in A-B, for cells cultured in IL-2 (left) or in B cell expansion media (right) at day 4 and 8. CD19+ cells are expanding only in the presence of B cell expansion media at the expense of CD4+ and CD8+ cells. (3D-3E) (3D) Spectral cytometry example and (3E) quantification of CD 19+ cells (B cells) for select tumor samples from Hepatocellular Carcinoma (HCC, red), Renal Cell Carcinoma (RCC, blue), or pancreatic cancer (Pancreas, orange) over time. Expansion of B lineage cells is evident from the frequency of CD19+ cells reaching a peak at day 7-12 and subsequent differentiation into antibody secreting cells (expected to be CD19-) in later stages of culture. (3F)Comparison of B lineage cell enrichment at late culture stages (red), as quantified by IgK+ cells (BCR+ cells, above) or CD3- cells (non-T cells, below) in culture, to cell cultures without enrichment (blue). B cell enrichment was performed at the indicated day by a dotted line on the graph and performed for a pancreas tumor sample (Pancreas, left) or HCC tumor sample (Liver, right). (3G) Cell counts in TIL-B cultures from an RCC sample (left) or viability (right) where IL-21 was supplemented to the B cell expansion media (IMNC) at day 8 (red line) and as compared to B cell expansion media without IL-21 supplementation (blue). Frequency ofB cells (CD19+, CD3-), T cells (CD3+, CD19-) or plasma cells (CD19-CD38+) in an HCC sample cultured in B cell expansion media supplemented with 0,10 or lOOng / ml of IL-21. In green, are TIL from the sample, cultured in IL-2.

[0066] Figures 4A-4I: Characterization of antibodies from TIL-B. (4A) Graph of RCC OMG antibodies’ binding to RCC tumor cell lines and primary tumor cells (TIL-B matched), HCC tumor cell lines and primary tumor cells (TIL-B unmatched), Healthy donor PBMC T cells (Healthy Tissue) and as compared to 2ndonly binding in RCC tumor cell lines and primary tumor cells (Control). **** = pv<0.0001, *** = pv<0.001, * = pv<0.05, ns = pv>0.05. Each dot represents a different donor to target cell combination. (4B) Same as in 4A but for HCC OMG antibodies derived from HCC tumors. (4C) Same as in 4A but for pancreatic tumor OMG antibodies. (4D) Dose response curve of RCC OMG antibodies binding to a primary RCC tumor line. Polyclonal binding was assessed by quantification of IgL+ IgK+ cells after staining by spectral cytometry. Binding of OMG antibodies from the RCC tumor (TIL-B, blue) to the RCC tumor line was compared to polyclonal antibodies derived from B cell enriched from PBMC culture binding to the same RCC tumor line (PBMC, dark grey). OMG antibodies or PBMC derived antibodies were also tested for binding to healthy PBMC derived T cells (TIL-B / PBMC, green or PBMC / PBMC, light grey, respectively). (4E) Graph of RCC OMG antibodies tested for binding to RCC tumor cell lines and primary tumor cells (TIL-B matched) and compared to plasma antibodies derived from RCC patients (Plasma) or to antibodies derived from PBMC of healthy individuals (Healthy individuals). (4F) Same as in 4E but for HCC OMGs and samples. (4G) Same as in 4E but for pancreas tumor OMGS and samples. 4E-4G and 4A-4C include the same data of OMG antibodies, but with different comparisons. (4H) Primary RCC tumor line killing experiment with OMG antibodies derived from an RCC patient. OMG antibodies (TIL-B, dark grey) or healthy donor PBMC antibodies (PBMC, light grey) were supplemented at the indicated concentration (x-axis) and primary NK cells were supplemented to the culture.Specific killing was quantified by target tumor dead cells as acquired in spectral cytometry. Standard deviation is indicated as representative of two independent experiments with different donor antibodies. ****=p<0.0001 Two-Way ANOVA for comparison between groups and **=p<0.01, *=p<0.05 for Two-Way ANOVA with Sidak’s multiple comparison test for each indicated concentration. (41) Line graph of OMG antibodies from HCC or RCC TIL-B cultures binding to tumor cells or to healthy cells. TIL-Bs were from HCC or RCC cancers. ** = pv<0.01 by two way ANOVA with Fisher LSD’s for tumor to healthy comparisons.

[0067] Figures 5A-5E: Characterization of malignant effusion derived B cells and antibodies. (5A) Bar graph of antibody expressing cell counts per samples from the malignant effusions from 7 different tumor types. (5B) Pie charts representing the total cells in a sample (numbers in the middle) and fraction of lymphocytes (CD19+ BCMA- CD3- B cells: blue, CD3- BCMA+ Plasmablasts / cells: light blue, CD19- BCMA- other cells: black) in pleural or ascites donor samples. (5C) Bar graphs of activated B cells (left) and the IgG and IgM status (right) of B cells from PBMC or ascites. (5D) Antibody dependent killing assay employing either antibodies derived from ascites of a Uterine Cervix adenocarcinoma patient, plasma cell culture of cells from the same sample, or healthy donor PBMC culture derived antibodies. Standard deviation represents technical replicates and specific killing is normalized to samples without NK cells. ####=p<0.0001 Two-Way ANOVA for comparison between groups and ****=p<0.0001, *=p<0.05 for Two-Way ANOVA with Sidak’s multiple comparison test to the Healthy PBMC culture-derived control, for each indicated concentration, and as shaded in the legend. (5E) Bar graphs of OMG antibodies’ binding to tumor cell lines and primary tumor cells of various cancers. *** = pv<0.001, * = pv<0.05, ns = pv>0.05. OMG antibodies were incubated at 10 ug / ml for 10 minutes at room temperature and stained with secondary antibody against human IgG, IgM, IgL and IgK. Each dot of the OMG column represents a different donor to target cell combination. Each dot of the control column represents a different target cell stained only with secondary antibody.

[0068] Figures 6A-6C: Production of OMG antibodies from malignant effusions. (6A) Cells are collected and cultured for the production of antibodies which are then utilized for the treatment of multiple individuals and multiple tumor types. (6B) OMG antibody production can be done in a closed system enabling sample collection from tumor associated fluids, growth of antibody producing cells, purification of OMG antibodies and packagingfor autologous or allogeneic infusion. Ascites or pleural effusions are collected in bags and lymphocytes, or enriched B lineage cells are cultured in closed systems containing B lineage expansion media. Following culture, antibodies secreted by the culture are collected, purified with or without enrichment for tumor binding and packaged for infusion in the same original patient, other patients with the same tumor type or patients with different tumor types. (6C) Bar graph of the isotype pattern from antibodies purified from ascites and from the supernatant of ascites derived B cells / antibody producing cells.

[0069] Figures 7A-7E: Pooling polyclonal antibodies from multiple donors improves tumor cell targeting and functionality. (7A-D) Bar graphs indicating the percentage of tumor cell binding as assessed by flow cytometry of OMG antibodies from a single donor or multiple donors with the same type of cancer for (7A) renal cancer, (7B) head and neck cancer, (7C) breast cancer and (7D) ovarian cancer. *=pv<0.05, **=pv<0.01, ***=pv<0.001, ****=pv<0.0001 for paired two-sided t-test. (7E) Line graph of specific cell killing of tumor cells from a breast cancer by OMG antibodies from a single donor or multiple donors in the presence of primary NK cells. Antibodies isolated from the culture of B cells derived from PBMCs of a healthy donor were used as the control. ####=p<0.0001 Two-Way ANOVA for comparison between groups and ****=p<0.0001, *=p<0.05 for Two-Way ANOVA with Sidak’s multiple comparison test to the Healthy PBMC culture-derived control, for each indicated concentration, and as shaded in the legend.

[0070] Figures 8A-8D: Schematics of OMEGA antibody generation. (8A). Engineering strategy to fuse polyclonal OMG antibodies with a payload on the heavy chain C-terminus of the antibodies. The transgene integrates in the last exon of a secreted heavy chain constant segment. (8B) Engineering strategy to fuse polyclonal OMG antibodies on the light chain C-terminus of the antibodies. This strategy enables that all isotypes secreted by the TIL-B cultures are targeted for engineering. This strategy may enable antibody multimerization commonly occurring with IgA and IgM isoforms. Either or both IgK and IgL light chain constants may be targeted. (8C) Engineering strategy to generate mispairing free, bi-specific antibodies. Early exons in a specific IgH isotype are targeted for transgene integration. The transgene codes for the rest of the targeted heavy chain, mutated to include a Knob, for a full light chain with an anti-CD3 VH and for an anti-CD3 VL fused to an heavy chain constant domain including a Hole. The Knob-in-Hole mutations enable heavy chain dimerization with minimal mispairing. The VH-CL and VL-CH fusions enable light chain pairing with minimal mispairing. The Knob and Hole mutation locations may be swapped. (8D)Engineering strategy to produce polyclonal bi-specific T cell engagers or similar molecules, devoid of full heavy chain constant segments. Here, the payload is integrated in the VH-CH intron of the IgH locus. The transgene includes a full CHI domain to enable pairing with the endogenous light chain and is fused with a linked to an anti-CD3 scFv domain. This enables secretion of low half-life bi-specific polyclonal T cell engagers that may be able to reach solid tumors more conveniently than full antibodies. In 8A-8D, stop codons are indicated as a red bar, exons as boxes, introns as lines. UTR = untranslated region, SD= splice donor, SA=splice acceptor. Alt.PolyA = alternative Polyadenylation site. CH= constant heavy. VH= variable heavy, VL / K = variable light chain lambda or kappa.

[0071] Figures 9A-9F: Characterization of OMEGA antibodies. (9 A) TIDE analysis measuring CRISPR cleavage for the editing strategy targeting the constant kappa light chain. Each dot represents an independent donor derived enriched B cells. (9B) Engineering rates of antibodies with the integration of a cassette coding for an anti-CD3 domain fused to a C-termini His tag. Antibody engineering rates were detected by Spectral Cytometry on polyclonal B lineage cells cultured in expansion media. RNP= Ribonucleoprotein, composed of the Cas9 protein and the specific gRNA electroporated into the cells. In some of the conditions, the electroporation also includes dsDNA coding for the template integrating into the IgK locus. Engineering antibody rates are quantified to the rates of cells positive for the His tag, coded into the transgene, amongst the cells positive for the IgK light chain. Only cells electroporated with both the RNP and the DNA donor template are positive for significant detection of the His tag. ** = pv <0.01, ns = pv>0.05. (9C) Quantification of the results from 9B but with the comparison of cells positive for the His tag in the CD 19+ IgK-cells (IgK-) compared to the CD 19+ IgK+ cells (IgK+). (9D-9F) Supernatants from engineered B cells were taken and analyzed for the capacity of engineered antibodies to bind to CD3+ T cells. (9D) Spectral Cytometry example of T cells labeled with the supernatants and secondary antibodies detecting both the His Tag present only on engineered antibodies and for Ig (as detected by IgK+ His+ cells). As control, supernatants from non-engineered antibodies (-RNP -DNA control) were utilized. (9E) Quantification of T cell binding by nonengineered antibodies (-RNP -DNA control) compared to engineered antibodies (+RNP +DNA). * = pv <0.05. (9F) Same as in 9E but including both comparisons with nonengineered antibodies and with enriched antibodies. Analysis of binding to the T cell culture was performed either on the whole population or on pre-gated CD4+ cells or CD8+ cells.

[0072] Figures 10A-C: (10A) Bar graph of the percentage of B cells which are engineered as detected by His+ cells in the IgK+ cell population. UT: untouched cells. RNP: ribonucleoprotein only electroporated cells. OMEGA: RNP electroporated and AAV transduced cells. (10B) Bar graph of the percentage of T cells bound by the engineered antibodies. Binding is detected by flow cytometry with anti-HIS (Miltenyi #130-119-820) and anti -human IgG (Biolegend #410706) antibodies. (10C) Scatter plots of flow cytometry data for engineered antibodies from HCC TIL-Bs binding to HuH7.5 (left) and HCC281024 (right). PBMC-B cell derived antibodies are used as a control.

[0073] Figures 11A-B: Bar graphs of (11 A) antibody engineering percentage and (11B) T cell binding by antibodies fused to OKT3 (#600) or TR66 (#603). *** = p <0.001, **** = p <0.0001.

[0074] Figures 12A-F: Bar graphs of (12A, 12D) antibody engineering percentage, ***=p<0.001, ****=p<0.0001 for one-way ANOVA with Tukey’s multiple comparison test (12B, 12E) T cell binding, **=p<0.01, ****=p<0.0001 for unpaired t-test with Welsh’s correction, and (12C, 12F) tumor specific retention by OMEGA antibodies engineered by the knob-in-holes (KiH) approach (12A-C) or the BiTE approach (12D-E).

[0075] Figure 13: Heat maps representing data of OMG antibody binding to various tumor lines. Each row represents a different tumor line. Columns represent either the secondary antibody control, monoclonal antibody binding (targeting either EGFR, GPC3, Her2 or Her3) or OMG antibody binding as detected by Protein L-biotin+streptavidin binding (Genscript #M00097, Milteny #130-106-786).DETAILED DESCRIPTION OF THE INVENTION

[0076] The present invention, in some embodiments, provides methods of treating cancer in a subject comprising receiving plasmablasts and / or plasma cells from a tumor or malignant effusion, culturing the received cells in culture media, isolating polyclonal antibodies and administering the antibodies to the subject, thereby treating cancer are provided. Compositions comprising polyclonal antibodies from B cells, plasmablasts and / or plasma cells derived from a tumor or malignant effusion are also provided as are methods of enriching tumor derived B cells, plasmablasts and / or plasma cells in culture.

[0077] The invention is based, at least in part, on the surprising finding that polyclonal antibodies derived from B cells directly cultured with cancer cells can be an effective broad anticancer treatment. Adoptive cell therapy, such as tumor infiltrating lymphocyte (TIL) therapy, makes use of the broad anticancer nature of a mixed population of cells. Such therapies have an advantage over monoclonal therapies due to the polyclonal nature of cancer, and result in fewer escaping cancer cells. However, cell-based therapies have numerous drawbacks. In contrast, polyclonal antibody therapies provide the broad anticancer effect without the problems inherent to cell therapies. Further, use of the new culture methods disclosed herein allows for the production of large quantities of polyclonal antibodies from tumors and malignant effusions.

[0078] The invention is neither a cell therapy nor a conventional antibody therapy which makes use of monoclonal antibodies. Rather the invention utilizes the supernatant from cell cultures, or the antibodies found in tumor fluids. The invention relies on polycl onality, which is avoided in modem antibody therapeutic approaches. Further, it leverages the specificity provided by human-derived samples, as opposed to immunization of animals, to produce antibodies that do not require modifications such as glycosylation, to be employed in a clinical setting. The broad anticancer specificity provided by the polyclonality of the antibodies can be enhanced by engineering the antibody genomic locus. In this way polyclonality is retained while enhancing anticancer activity.

[0079] By a first aspect, there is provided a composition comprising polyclonal antibodies harvested from B cells, plasmablasts and / or plasma cells derived from a cancer.

[0080] By another aspect, there is provided a method of producing an anticancer therapeutic, the method comprising:a. receiving B cells, plasmablasts and / or plasma cells from a cancer; b. culturing the received plasmablasts and / or plasma cells in culture media;andc. isolating a composition of polyclonal antibodies from the culture media; thereby producing an anticancer therapeutic.

[0081] By another aspect, there is provided a method of producing an anticancer therapeutic, the method comprising:a. receiving a malignant effusion from a subject suffering from cancer;b. isolating polyclonal antibodies from the received malignant effusion; and c. producing a composition comprise the isolated polyclonal antibodies; thereby producing an anticancer therapeutic.

[0082] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, excipient or adjuvant. As used herein, the term “carrier,” “excipient,” or “adjuvant” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S.Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelies, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0083] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0084] In some embodiments, the polyclonal antibodies are harvested from B cells. In some embodiments, the polyclonal antibodies are harvested from plasmablasts. In some embodiments, the polyclonal antibodies are harvested from plasma cells. In some embodiments, the polyclonal antibodies are harvested from plasmablasts and plasma cells. In some embodiments, B cells are received. In some embodiments, plasmablasts are received. In some embodiments, plasma cells are received. In some embodiments, the B cells, plasmablasts and / or plasma cells are derived from a tumor. In some embodiments, the B cells, plasmablasts and / or plasma cells are derived from a malignant effusion. In some embodiments, the B cells, plasmablasts and / or plasma cells are in culture. In someembodiments, the polyclonal antibodies are derived from cells in culture. In some embodiments, the polyclonal antibodies have been purified, isolated or extracted from culture.

[0085] In some embodiments, from a cancer is from a tumor. In some embodiments, from a cancer is from a malignant effusion. In some embodiments, from a cancer is from a tumor or a malignant effusion. A malignant effusion refers to the accumulation of fluid in a body cavity due to the presence of cancer. The effusion itself contains cancerous cells as well as immune cells. Such effusions can occur in various parts of the body including the pleural cavity (pleural effusion), peritoneal cavity (ascites), or pericardial cavity (pericardial effusion). In some embodiments, the malignant effusion is a pleural effusion. In some embodiments, the malignant effusion is an ascites. In some embodiments, the malignant effusion is selected from a pleural effusion and an ascites.

[0086] In some embodiments, the culture comprises cancer cells. In some embodiments, the cancer cells are tumor cells. In some embodiments, the cancer cells are cancer cells from the effusion. In some embodiments, the culture comprises non-B cell immune cells. In some embodiments, the culture comprises T cells. In some embodiments, T cells are CD3 positive cells. In some embodiments, T cells are CD4 T cells. In some embodiments, T cells are CD8 T cells.

[0087] In some embodiments, the composition is a non-naturally occurring composition. In some embodiments, the polyclonal antibodies are different than polyclonal antibodies produced in vivo. In some embodiments, the polyclonal antibodies are engineered. In some embodiments, the carrier, excipient or adjuvant comprises a non-naturally occurring component. In some embodiments, a non-naturally occurring component is an inorganic component. In some embodiments, the non-naturally occurring component is a supplement.

[0088] In some embodiments, the composition comprises cells. In some embodiments, the composition is devoid of cells. In some embodiments, cells are tumor cells. In some embodiments, cells are immune cells. In some embodiments, immune cells are B cells. In some embodiments, immune cells are plasmablasts. In some embodiments, immune cells are plasma cells. In some embodiments, immune cells are plasmablasts and plasma cells. In some embodiments, the composition consists of the polyclonal antibodies. In some embodiments, the composition consists of the polyclonal antibodies and the pharmaceutically acceptable carrier, excipient or adjuvant.

[0089] In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for intratumoral administration. In some embodiments, the pharmaceutical composition is for use in treating cancer. In some embodiments, the pharmaceutical composition is for use in the production of a medicament for treating cancer.

[0090] In some embodiments, the method further comprises administering the composition to a subject. In some embodiments, the method is a method of producing an anticancer agent and treating a subject. In some embodiments, the method is a method of treating a subject. In some embodiments, a composition of polyclonal antibodies is administered. In some embodiments, polyclonal antibodies derived from at least one subject suffering from cancer are administered. In some embodiments, polyclonal antibodies derived from a plurality of subjects suffering from cancer are administered. In some embodiments, polyclonal antibodies from at least two different subjects are administered. In some embodiments, polyclonal antibodies from at least two different subjects are pooled and the pool polyclonal antibodies are administered. In some embodiments, at least two is a plurality. As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include parenteral, subcutaneous, oral, intramuscular, intratumoral or intraperitoneal.

[0091] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0092] In some embodiments, the subject suffers from cancer. In some embodiments, the subject is in need of a method of the invention. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is treated. In some embodiments, the subject is administered the composition. In some embodiments, the composition is a composition of the invention.

[0093] In some embodiments, the subject provides a tumor sample. In some embodiments, the subject provides a malignant effusion sample. In some embodiments, a tumor sample is provided from a subject suffering from cancer. In some embodiments, a malignant effusion sample is provided from a subject suffering from cancer. In some embodiments, the cancer is from a subject suffering from cancer. In some embodiments, the subject suffering from cancer is the subject in need of treatment. In some embodiments, the subject suffering from cancer is not the subject in need of treatment. In some embodiments, the B cells, plasmabalasts and / or plasma cells are from a subject suffering from cancer that is not the subject in need of treatment. In some embodiments, in need of treatment is in need of treatment by a method of the invention. In some embodiments, the cancer in the subject in need of treatment is the same type of cancer as the cancer in the subject suffering from cancer. In some embodiments, the cancer in the subject in need of treatment is the same type of cancer as the cancer cells in the sample. In some embodiments, the received cells are from a subject suffering from a cancer of the same type as the cancer of the subject in need of treatment. In some embodiments, the cancer in the subject in need of treatment is the same type of cancer as the tumor. In some embodiments, the cancer in the subject in need of treatment is the same type of cancer as the cancer that produced the malignant effusion. In some embodiments, the cancer in the subject in need of treatment is the same type of cancer as the cancer cells in the malignant effusion. In some embodiments, the same type of cancer is cancer of the same tissue, organ or cell type. In some embodiments, the same type of cancer is cancer of the same tissue. In some embodiments, the same type of cancer is cancer of the same organ. In some embodiments, the same type of cancer is cancer of the same tissue cell type. In some embodiments, the B cells, plasmablasts and / or plasma cells are from the sample. In some embodiments, from the sample is derived from the sample. In some embodiments, the plasmablasts and / or plasma cells are derived from B cells from the sample.

[0094] In some embodiments, cancer is a proliferative disease. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a carcinoma. In some embodiments, the carcinoma is an adenocarcinoma. In some embodiments, the cancer is selected from hepato-biliary cancer, liver cancer, cervical cancer, urogenital cancer (e.g., urothelial cancer), testicular cancer, prostate cancer, thyroid cancer, ovarian cancer, nervous system cancer, ocular cancer, lung cancer, soft tissue cancer, bone cancer, pancreatic cancer, bladder cancer, brain cancer, skin cancer (e.g., melanoma), intestinal cancer, hepatic cancer, liver cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer,gastroesophageal cancer, gastrointestinal cancer, breast cancer (e.g., triple negative breast cancer), renal cancer (e.g., renal carcinoma), uterine cancer and head and neck cancer. In some embodiments, the cancer is selected from renal cancer, liver cancer, pancreatic cancer, lung cancer, gastric cancer, ovarian cancer, skin cancer, head and neck cancer, uterine cancer and cervical cancer. In some embodiments, the cancer is selected from renal cancer, liver cancer, pancreatic cancer, lung cancer, gastric cancer, uterine cancer and cervical cancer. In some embodiments, the cancer is selected from renal cancer, liver cancer, pancreatic cancer, lung cancer, gastric cancer, and uterine or cervical cancer. In some embodiments, the cancer is selected from renal cancer, liver cancer, pancreatic cancer, lung cancer, gastric / gastrointestinal cancer, ovarian cancer, breast cancer, skin cancer, head and neck cancer, uterine cancer, brain cancer, bladder cancer, and cervical cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the renal cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is uterine cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is uterine or cervical cancer. In some embodiments, uterine or cervical cancer is uterine cervix adenocarcinoma. In some embodiments, the cancer is selected from renal cancer, liver cancer, pancreatic cancer and uterine / cervical cancer. In some embodiments, the cancer is selected from renal cancer, liver cancer, and pancreatic cancer. In some embodiments, a type of cancer is a cancer with the same tissue / cell type of origin.

[0095] B cells are well known in the art and refer to B lymphocytes, a type of white blood cell. In some embodiments, B cells are CD19 positive. In some embodiments, B cells are CD20 positive. In some embodiments, B cells are CD21 positive. Both mature B cells and activated B cells are positive for CD 19 and so CD 19 is most commonly used as the marker for B cells. In some embodiments, a CD 19 positive (CD 19+) population of cells is a B cell population. B cells express the B cell receptor (BCR) which is membrane bound but whencleaved is essentially equivalent to an antibody. While B cells do secrete low levels of antibodies, this is not their major function.

[0096] Plasmablasts are well known in the art and refer to an intermediate stage in the differentiation of activated B cells into plasma cells. Plasmablasts secrete antibodies in greater amounts that B cells, but not as efficiently as plasma cells. A plasmablast is derived from a B cell that has been contacted by an antigen and each plasmablast produces antibodies specific to that antigen. A population of plasmablasts derived from B cells that have been exposed to cancer cells will produce a variety of antibodies against a variety of antigens and so the antibodies produced will be polyclonal in nature. In some embodiments, a plasmablast is CD138 positive. Plasmablasts can be CD138 positive, but some plasmablasts are negative for CD138. In some embodiments, plasmablasts are CD38 positive. In some embodiments, plasmablasts are CD 19 positive. In some embodiments, plasmablasts are CD21 positive. In some embodiments, plasmablasts are CD20 positive but at lower levels than in B cells. In some embodiments, plasmablasts are B-cell maturation antigen (BCMA / TBFRSF17) negative. In some embodiments, plasmablasts are CD38 positive and BCMA negative. In some embodiments, plasmablasts are CD19 positive, CD38 positive and BCMA negative.

[0097] Plasma cells are well known in the art and refer to fully differentiated cells of the B cell lineage. Plasma cells are the main antibody producing / secreting cell and secrete antibodies in greater numbers and more efficiently than plasmablasts. Since plasma cells derived from plasmablasts, which derived from a B cell that has been contacted by an antigen, each plasma cell produces antibodies specific to that antigen. A population of plasma cells derived from B cells that have been exposed to cancer cells will produce a variety of antibodies against a variety of antigens and so the antibodies produced will be polyclonal in nature. In some embodiments, plasma cells are CD138 positive. In some embodiments, plasma cells are CD38 positive. In some embodiments, plasma cells are CD 19 negative. In some embodiments, plasma cells are CD20 negative. In some embodiments, plasma cells are BCMA positive. In some embodiments, plasma cells are CD138 positive and BCMA positive. In some embodiments, plasma cells are CD38 positive and BCMA positive. In some embodiments, plasma cells are CD38 positive, CD138 positive and BCMA positive. In some embodiments, plasma cells are CD 19 negative, CD38 positive and BCMA positive. In some embodiments, plasma cells are CD 19 negative and BCMA positive. In some embodiments, plasma cells are CD 19 negative and CD38 positive. In someembodiments, plasma cells are CD 19 negative and CD138 positive. In some embodiments, plasma cells are CD 19 negative, CD138 positive, CD38 positive and BCMA positive.

[0098] In some embodiments, receiving is receiving B cells from the cancer. In some embodiments, receiving is receiving B cells from a malignant effusion. In some embodiments, receiving is receiving plasmablasts from the cancer. In some embodiments, receiving is receiving plasmablasts from a malignant effusion. In some embodiments, receiving is receiving plasma cells from the cancer. In some embodiments, receiving is receiving plasma cells from a malignant effusion. In some embodiments, receiving is receiving a mixed population of cells from the cancer. In some embodiments, receiving is receiving a mixed population of cells from a malignant effusion. In some embodiments, the mixed population is a mixed population of immune cells. In some embodiments, the mixed population comprises cancer cells. In some embodiments, immune cells comprise T cells and B cells. In some embodiments, immune cells comprise T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages and monocytes. In some embodiments, immune cells are lymphocytes.

[0099] In some embodiments, the receiving comprises receiving a sample. In some embodiments, the sample is from the cancer. In some embodiments, the sample comprises immune cells. In some embodiments, the sample comprises cancer cells. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a malignant effusion sample. In some embodiments, the receiving further comprises isolating B cells from the received sample. In some embodiments, the receiving further comprises isolating CD 19 positive cells from the received sample. In some embodiments, the sample is a malignant effusion sample. In some embodiments, the receiving further comprises isolating plasmablasts from the received sample. In some embodiments, the receiving further comprises isolating plasma cells from the received sample. In some embodiments, the receiving further comprises isolating B cells, plasmablasts and / or plasma cells from the received sample. In some embodiments, the receiving further comprises isolating B cells, plasmablasts and plasma cells from the received sample. In some embodiments, the receiving further comprises isolating cells positive for syndecan 1 (CD138 / SDC1), B-cell maturation antigen (BCMA / TBFRSF17), CD38 or a combination thereof from said sample. In some embodiments, the receiving further comprises isolating cells positive for CD 19, syndecan 1 (CD138 / SDC1), B-cell maturation antigen (BCMA / TBFRSF17), CD38 or a combination thereof from said sample. In some embodiments, the receiving furthercomprises isolating cells positive for CD 19, CD138 or CD38 and BCMA from the sample. Surface markers for the various cell types are known in the art and isolation of these cell types can be performed for example using commercially available kits (e.g., Miltenyi kits #130-091-151 and # #130-093-628) or by FACS sorting.

[0100] In some embodiments, isolating is purifying. In some embodiments, isolating is enriching. In some embodiments, an isolated population comprises at least 30% purity. In some embodiments, an isolated population comprises at least 50% purity. In some embodiments, an isolated population comprises at least 70% purity. In some embodiments, an isolated population comprises at least 80% purity. In some embodiments, an isolated population comprises at least 85% purity. In some embodiments, an isolated population comprises at least 90% purity. In some embodiments, an isolated population comprises at least 95% purity.

[0101] In some embodiments, the receiving further comprises isolating cancerous cells from the received sample. In some embodiments, the isolated B cells are cultured with the isolated cancerous cells. In some embodiments, the isolated B cells and plasmablasts / plasma cells are cultured with the isolated cancerous cells. In some embodiments, B cells are isolated after culturing with cancer cells. In some embodiments, plasmablasts and / or plasma cells are isolated after culturing with cancer cells. In some embodiments, B cells, plasmablasts and plasma cells are isolated after culturing with cancer cells.

[0102] In some embodiments, the culturing is in media. In some embodiments, the media is B cell media. In some embodiments, the media is B cell expansion media. In some embodiments, the culturing is to enrich for plasmablasts and / or plasma cells. In some embodiments, the culturing is for a time sufficient to enrich for plasmablasts and / or plasma cells. In some embodiments, immune cells are isolated and cultured in media for a time sufficient to enrich for B cells. In some embodiments, immune cells are isolated and cultured in media for a time sufficient to enrich for plasmablasts and / or plasma cells. In some embodiments, immune cells are isolated and cultured in media for a time sufficient to enrich for B cells, plasmablasts and / or plasma cells. In some embodiments, immune cells are isolated from the cancer, cultured in media from a time sufficient to enrich for B cells, the enriched B cells are isolated and the isolated B cells are cultured to enrich for plasmablasts and / or plasma cells. In some embodiments, T cells are depleted from the immune cells before the culturing. Negative selection against CD3 positive cells can be achieved using T cell isolation kits (such as from Miltenyi) or by FACS sorting, as non-limiting examples.

[0103] In some embodiments, the media is B cell culture media. In some embodiments, the media is B cell expansion media. In some embodiments, the B cell expansion media is Immunocult Human B cell expansion media. Commercially available B cell media is well known and can be purchases for example from companies such as R&D Systems, Thermo Fisher Scientific, Miltenyi Biotech, 3H Biomedical and StemCell Technologies to name but a few. In some embodiments, the media is RPMI. In some embodiments, RPMI is RPMI 1640. In some embodiments, the media is supplemented. In some embodiments, the supplement is fetal bovine serum (FBS). In some embodiments, the FBS is heat inactivated. In some embodiments, the media is chemically defined media. In some embodiments, the supplement is IL-21. In some embodiments, the supplement is beta mercaptoethanol. In some embodiments, the supplement is RP 105 / CD 180. In some embodiments, the supplement is IL-7. In some embodiments, the supplement is IL-4. In some embodiments, the supplement is CD40 ligand (CD40L). In some embodiments, CD40L is meagCD40L. In some embodiments, the supplement is an oligodeoxyribonucleotide containing CpG motifs. In some embodiments, the oligodeoxyribonucleotide is CpG ODN2006. In some embodiments, the supplement is IL- 10. In some embodiments, the supplement is IL-2. In some embodiments, the supplement is IL-15. In some embodiments, the supplement is IL-6. In some embodiments, the supplement is B-cell activating factor (BAFF / TNFSF13B). In some embodiments, the supplement is HEPES. In some embodiments, the supplement is R848 (a Toll-like receptor 7 and 8 agonist). In some embodiments, the supplement is gentamicin.

[0104] In some embodiments, the media is Immunocult Human B cell expansion media supplemented with IL-2L In some embodiments, the media is RPMI supplemented with FBS, beta-mercaptoethanol, RP105 and IL-7. In some embodiments, the media is RPMI supplemented with FBS, beta-mercaptoethanol, RP105 and IL-4. In some embodiments, the media is RPMI supplemented with FBS, beta-mercaptoethanol, RP105, IL-4 and IL-21. In some embodiments, the media is RPMI supplemented with FBS, beta-mercaptoethanol, CD40L, oligodeoxyribonucleotides containing CpG motifs, IL-10, IL-2, IL-15 and IL-6. In some embodiments, the media is RPMI supplemented with FBS, beta-mercaptoethanol, CD40L, IL-4, IL-21, and BAFF. In some embodiments, the media is RPMI supplemented with FBS, beta-mercaptoethanol, In some embodiments, the media is RPMI supplemented with FBS, beta-mercaptoethanol, CD40L and IL4.

[0105] In some embodiments, FBS is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% FBS. Each possibility represents a separate embodiment of the invention. In some embodiments, FBS is about 10% FBS. In some embodiments, beta-mercaptoethanol is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 uM beta-mercaptoethanol. Each possibility represents a separate embodiment of the invention. In some embodiments, beta-mercaptoethanol is about 50 uM beta-mercaptoethanol. In some embodiments, RP105 is about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 ug / ml RP105. Each possibility represents a separate embodiment of the invention. In some embodiments, RP105 is about 2 ug / ml RP105. In some embodiments, IL-7 is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / ml IL-7. Each possibility represents a separate embodiment of the invention. In some embodiments, IL-7 is about 10 ng / ml IL-7. In some embodiments, IL-4 is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / ml IL-4. Each possibility represents a separate embodiment of the invention. In some embodiments, IL-4 is about 10 ng / ml IL-4. In some embodiments, IL-21 is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ng / ml IL-21. Each possibility represents a separate embodiment of the invention. In some embodiments, IL-21 is about 100 ng / ml IL-21. In some embodiments, CD40L (e.g., megaCD40L) is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ng / ml CD40L. Each possibility represents a separate embodiment of the invention. In some embodiments, CD40L (e.g., megaCD40L) is about 100 ng / ml CD40L. In some embodiments, CpG ODN2006 is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ug / ml CpG ODN2006. Each possibility represents a separate embodiment of the invention. In some embodiments, CpG ODN2006 is about 1 ug / ml CpG ODN2006. In some embodiments, IL-10 is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 ng / ml IL- 10. Each possibility represents a separate embodiment of the invention. In some embodiments, IL- 10 is about 50 ng / ml IL- 10. In some embodiments, IL-2 is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 ng / ml IL-2. Each possibility represents a separate embodiment of the invention. In some embodiments, IL-2 is about 50 ng / ml IL-2. In some embodiments, IL-15 is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / ml IL -15. Each possibility represents a separate embodiment of the invention. In some embodiments, IL-15 is about 10 ng / ml IL-15. In some embodiments, IL-6 is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, or 500 ng / ml IL-6. Each possibility represents a separate embodiment of the invention. In some embodiments, IL-6 is about 50 ng / ml IL-6. In some embodiments, BAFF is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ug / ml BAFF. Each possibility represents a separate embodiment of the invention. In some embodiments, BAFF is about 1 ug / ml BAFF. In some embodiments, HEPES is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 mM HEPES. Each possibility represents a separate embodiment of the invention. In some embodiments, HEPES is about 5 mM betamercaptoethanol. In some embodiments, R848 is about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 ug / ml R848. Each possibility represents a separate embodiment of the invention. In some embodiments, R848 is about 1 ug / ml R848. In some embodiments, gentamicin is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 gg / ml gentamicin. Each possibility represents a separate embodiment of the invention. In some embodiments, gentamicin is about 50 ug / ml gentamicin.

[0106] In some embodiments, a time sufficient to enrich or expand B cells is at least 3, 4, 5, 6, 7, 8, 9, or 10 days. Each possibility represents a separate embodiment of the invention. In some embodiments, a time sufficient to enrich or expand B cells is at least 5 days. In some embodiments, a time sufficient to enrich or expand B cells is at least 7 days. In some embodiments, a time sufficient to enrich or expand B cells is at most 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. Each possibility represents a separate embodiment of the invention. In some embodiments, a time sufficient to enrich or expand B cells is at most 12 days. In some embodiments, a time sufficient to enrich or expand B cells is at most 15 days. In some embodiments, a time sufficient to enrich or expand B cells is 7-12 days. In some embodiments, a time sufficient to enrich or expand B cells is 7-15 days. In some embodiments, a time sufficient to enrich or expand B cells is 5-12 days. In some embodiments, a time sufficient to enrich or expand B cells is 5-15 days.

[0107] In some embodiments, after B cells are expanded / enriched they are isolated and then are further cultured to produce plasmablasts and plasma cells. In some embodiments, the isolating occurs after the sufficient time. In some embodiments, the isolating occurs on about day 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of culturing. Each possibility represents a separate embodiment of the invention. In some embodiments, the isolating occurs on about day 7 of culturing. In some embodiments, the isolating occurs on about day 10 of culturing. In some embodiments, the isolating occurs on about day 12 of culturing. In some embodiments, theculturing after the B cell isolation is at least 3, 4, 5, 6, 7, 8, 9 or 10 days. Each possibility represents a separate embodiment of the invention. In some embodiments, the culturing after the B cell isolation is at least 3 days. In some embodiments, the culturing after the B cell isolation is at least 5 days. In some embodiments, the culturing after the B cell isolation is at least 7 days.

[0108] In some embodiments, the method does not comprise a step of enriching for tumor necrosis factor ligand superfamily member 9 (TNFSF9 / 4-1BBL) positive cells. In some embodiments, the method does not comprise a step of selecting TNFSF9+ cells. Methods of producing a cell -therapy often require selecting 4-1BBL positive cells, however, as the current therapeutic is not a cell therapy, but rather a polyclonal antibody therapy there is no need (and indeed it is detrimental) to select / enrich for such cells. In some embodiments, the method does not comprise a step of immortalizing cells. In some embodiments, the immune cells are not immortalized. In some embodiments, the method does not comprise a step of isolating antibodies from immortalized cells. In some embodiments, a method that does not comprise is a method that is devoid of.

[0109] In some embodiments, the method is performed on received cells from a plurality of subjects. In some embodiments, the polyclonal antibodies are from a plurality of subject’s malignant effusions. In some embodiments, steps of the method are performed on cells form a plurality of subjects. In some embodiments, the steps are steps a-b. In some embodiments, the steps are steps a-c. In some embodiments, the plurality is at least 2 subjects. In some embodiments, the plurality is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45 or 50 subjects. Each possibility represents a separate embodiment of the invention. In some embodiments, the plurality is at least 5 subjects. In some embodiments, the plurality of subjects all suffer from the same type of cancer. In some embodiments, the plurality of subjects suffer from the same type of cancer as the subject in need of treatment. In some embodiments, a type of cancer is a cancer with the same tissue / cell type of origin. In some embodiments, the subjects of the plurality are different subjects. In some embodiments, cells from at least two different subjects suffering from cancer are received, the cells from the at least two different subjects are cultured, polyclonal antibodies from the culture of the cells from at least two different subjects are isolated and the isolated polyclonal antibodies which are derived from two different subjects are administered to the subject in need of treatment. In some embodiments, at least two is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30,35, 40, 45 or 50 subjects. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 2 is at least 5.

[0110] In some embodiments, the method further comprises engineering the B cells. In some embodiments, the method further comprises engineering the plasmablasts. In some embodiments, the method further comprises engineering the plasma cells. In some embodiments, the method further comprises engineering the antibodies. In some embodiments, the engineering is genetic engineering. In some embodiments, the engineering is done while the cells are in culture. Methods of genetic engineering are well known in the art and include, but are not limited to RNA-guided nuclease engineering (e.g., CRISPR), site-directed mutagenesis, recombinant engineering, and viral transduction engineering. Any method known in the art may be used. In some embodiments, the genetic engineering comprises RNA-guided nuclease engineering. Examples of RNA-guided nuclease engineering include CRISPR-Cas, meganuclease, zinc finger nuclease and TALE nucleases to name but a few. In some embodiments, the RNA-guided nuclease engineering is CRISPR. In some embodiments, CRISPR is CRISPR-Cas engineering. In some embodiments, Cas is Cas9. In some embodiments, the genetic engineering comprises expressing an exogenous nucleic acid in the cells. In some embodiments, expression is incorporating the exogenous nucleic acid into the genome of the cells. In some embodiments, the genetic engineering comprises expressing an exogenous protein in the cells. In some embodiments, into the genome of the cells is into an endogenous antibody locus. In some embodiments, into the genome of the cells is into an endogenous coding region of an antibody.

[0111] In some embodiments, the exogenous protein is an immune effector. In some embodiments, the immune effector is a cytokine. In some embodiments, the cytokine is a proinflammatory cytokine. In some embodiments, the cytokine is selected from IL-21, IL-2, IL-15, IL-7, IL-1, IL-6, TNFA, IL-8, IL-12, IL-17, IL-18, and IFNG. In some embodiments, the cytokine is selected from IL-21, IL-2, IL- 15, and IL-7. In some embodiments, the cytokine is IL-21. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-15. In some embodiments, the cytokine is IL-7.

[0112] In some embodiments, the exogenous protein is a T cell engager. In some embodiments, the immune effector is a T cell engager. In some embodiments, the genetic engineering comprises expression a T cell engager in the cells. In some embodiments, a T cell engager is a protein or protein complex that binds to T cells. In some embodiments, the T cell engager is an anti-CD3 protein or protein complex. In some embodiments, the T cellengager is an anti-CD3 single chain antibody (scFv). In some embodiments, the T cell engager is an anti-CD3 single domain antibody. In some embodiments, a single domain antibody is a camelid or shark antibody. In some embodiments, a single domain antibody is a camelid antibody (i.e., a VHH). In some embodiments, the T cell engager is an anti-CD3 antibody.

[0113] In some embodiments, the genetic engineering comprises linking the exogenous protein to an endogenous antibody. In some embodiments, the genetic engineering comprises linking the exogenous protein to an endogenous heavy chain of an antibody. In some embodiments, the genetic engineering comprises linking the exogenous protein to an endogenous light chain of an antibody. In some embodiments, the heavy chain is a heavy chain constant region. In some embodiments, a light chain is a light chain constant region. In some embodiments, the linking is producing a bi-specific antibody. In some embodiments, the engineered antibody is a bispecific antibody. In some embodiments, the specificity is to an immune protein and the cancer target antigen which activated the B cell. It will be understood that the engineered antibodies will also be polyclonal. A population of B cells, plasmablasts and / or plasma cells will be modified. Each cell with have antibodies against a different cancer antigen depending on which antigen activated the original B cell and will also have an anti-immune protein antibody or antigen binding fragment thereof. Thus, there will be produced a plurality of bispecific antibodies that are polyclonal.

[0114] In some embodiments, the genetic engineering comprises linking the T cell engager to an endogenous antibody. In some embodiments, the genetic engineering comprises linking the T cell engager to an endogenous heavy chain of an antibody. In some embodiments, the genetic engineering comprises linking the T cell engager to an endogenous light chain of an antibody. In some embodiments, the heavy chain is a heavy chain constant region. In some embodiments, a light chain is a light chain constant region. In some embodiments, the linking is producing a bi-specific antibody. In some embodiments, the engineered antibody is a bispecific antibody. In some embodiments, the specificity is to CD3 and the cancer target antigen which activated the B cell. It will be understood that the engineered antibodies will also be polyclonal. A population of B cells, plasmablasts and / or plasma cells will be modified. Each cell with have antibodies against a different cancer antigen depending on which antigen activated the original B cell and will also have an anti-CD3. Thus, there will be produced a plurality of bispecific antibodies that are polyclonal.

[0115] Anti-CD3 antibodies, single chain antibodies and single domain antibodies are well known in the art and any such may be used. Example of such antibodies include OKT3 and TR66. In some embodiments, the anti-CD3 single chain antibody comprise AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLES GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTM NWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRA EDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 1). SEQ ID NO: 1 is exemplified hereinbelow in the examples section. In some embodiments, the anti-CD3 single chain antibody consists of SEQ ID NO: 1. In some embodiments, the engineering comprises expressing or integrating into the cell a nucleic acid molecule that encodes SEQ ID NO: 1. In some embodiments, the anti-CD3 single chain antibody comprise QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPS RGYTNYNQKFKDKATLTTDKS S ST AYMQLS SLTSEDS AVYYC ARYYDDHYCLD Y WGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKV TMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTIS GMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 2). SEQ ID NO: 2 is OKT3. In some embodiments, the anti-CD3 single chain antibody consists of SEQ ID NO: 2. In some embodiments, the engineering comprises expressing or integrating into the cell a nucleic acid molecule that encodes SEQ ID NO: 2. In some embodiments, the anti-CD3 single chain antibody comprise QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPS RGYTNYNQKFKDKATLTTDKS S ST AYMQLS SLTSEDS AVYYC ARYYDDHYSLDY WGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKV TMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTIS SMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 3). SEQ ID NO: 3 is TR66. In some embodiments, the anti-CD3 single chain antibody consists of SEQ ID NO: 3. In some embodiments, the engineering comprises expressing or integrating into the cell a nucleic acid molecule that encodes SEQ ID NO:3. In some embodiments, the T cell engager is selected from SEQ ID NO: 1-3. In some embodiments, engineering comprises expressing or integrating into the cell a nucleic acid molecule that encodes a T cell engager selected from SEQ ID NO: 1-3.

[0116] In some embodiments, the exogenous protein is selected from a T cell engager, a cytokine, a chemokine, and an anti-immune protein antibody or antigen binding fragment thereof. In some embodiments, the exogenous protein is a T cell engager. In some embodiments, the exogenous protein is a cytokine. In some embodiments, the exogenous protein is a chemokine. In some embodiments, the exogenous protein is a cytokine or chemokine. In some embodiments, the exogenous protein is an anti-immune protein antibody or antigen binding fragment thereof.

[0117] In some embodiments, the engineering comprises fusing to an endogenous antibody. In some embodiments, fusing to an endogenous antibody comprises fusing to an endogenous light chain. In some embodiments, fusing to an endogenous antibody comprises fusing to an endogenous heavy chain. In some embodiments, the fusion is to the terminus of the chain. In some embodiments, the terminus is the C-terminus. In some embodiments, the fusion is to the constant region. In some embodiments, to the constant region is within the constant region. In some embodiments, the engineering follows the scheme presented in Figure 8A.It will be understood that while Figure 8A shows a CD3 engager, the use of any exogenous protein is envisioned and encompassed in the scope of the invention. In some embodiments, the engineering follows the scheme presented in Figure 8B. It will be understood that while Figure 8B shows a CD3 engager, the use of any exogenous protein is envisioned and encompassed in the scope of the invention.

[0118] In some embodiments, an exogenous antibody is fused. In some embodiments, an exogenous single chain antibody is fused. In some embodiments, an exogenous single domain antibody is fused. In some embodiments, an exogenous cytokine is fused. In some embodiments, an exogenous chemokine is fused. In some embodiments, the immune effector is an antibody, single chain antibody or single domain antibody. In some embodiments, the antibody is an immune checkpoint inhibitor (ICI). In some embodiments, the antibody is a full IgG. In some embodiments, the antibody is a single chain antibody. In some embodiments, the antibody is a single domain antibody. In some embodiments, the antibody is anti-CD3. In some embodiments, the antibody is anti-CD28. In some embodiments, the antibody is anti-CD40. In some embodiments, the antibody is anti- Programmed cell death protein 1 (PD1). In some embodiments, the antibody is anti- Programmed death-ligand 1 (PD-L1). In some embodiments, the antibody is anti- Cytotoxic T-lymphocyte associated protein 4 (CTLA4). In some embodiments, the antibody is anti- C-X-C chemokine receptor type 5 (CXCR5). In some embodiments, the antibody is anti-CXCR4. In some embodiments,the checkpoint protein is selected from PD-1, PD-L1 and CTLA4. In some embodiments, the cytokine or chemokine is selected from interleukin 15 (IL-15), IL-7, IL-2, IL-21, IL-12, IL-4, C-X-C motif chemokine 12 (CXCL12), and CXCL13. In some embodiments, the cytokine or chemokine is IL-15, IL-7, IL-2, IL-21, IL-12, IL-4, CXCL12, or CXCL13. Each possibility represents a separate embodiment of the invention.

[0119] The various interleukins and generally proinflammatory cytokines are well known in the art and the amino acid sequences of these proteins and the nucleotide sequences encoding those amino acid sequences are also known. The human IL-15 gene sequence can be found at Entrez gene ID 3600 and the protein sequence can be found at UniProt ID P40933. The human IL-7 gene sequence can be found at Entrez gene ID 3574 and the protein sequence can be found at UniProt ID P13232. The human IL-2 gene sequence can be found at Entrez gene ID 3558 and the protein sequence can be found at UniProt ID P60568. The human IL-21 gene sequence can be found at Entrez gene ID 59067 and the protein sequence can be found at UniProt ID Q9HBE4. In some embodiments, IL-12 is IL-12a or IL-12b. The human IL-12a gene sequence can be found at Entrez gene ID 3592 and the protein sequence can be found at UniProt ID P29459. The human IL-12b gene sequence can be found at Entrez gene ID 3593 and the protein sequence can be found at UniProt ID P29460. The human IL-4 gene sequence can be found at Entrez gene ID 3565 and the protein sequence can be found at UniProt ID P05112. The human CXCL12 (also known as Stromal cell-derived factor 1; SDF1) gene sequence can be found at Entrez gene ID 6387 and the protein sequence can be found at UniProt ID P48061. The human CXCL13 (also known as B lymphocyte chemoattractant; BLC or B cell -attracting chemokine 1; BCA1) gene sequence can be found at Entrez gene ID 10563 and the protein sequence can be found at UniProt ID 043927.

[0120] Therapeutic antibodies that bind CD-3 are well known and include for example muromonab, and teplizumab. Therapeutic antibodies that bind CD-28 are well known and include for example theralizumab, and FR104 / VEL-101. Therapeutic antibodies that bind PD-1 are well known and include for example pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, tislelizumab, penpulimab, sintilimab, and serplulimab. Therapeutic antibodies that bind PD-L1 are well known and include for example atezolizumab, durvalumab, avelumab, cosibelimab, envafolimab, adebrelimab, and sugemalimab. Therapeutic antibodies that bind CTLA4 are well known and include for example ipilimumab and tremelimumab. Therapeutic antibodies that bind CXCR4 are well known and include for example ulocuplumab. Therapeutic antibodies that bind CXCR5 arewell known and include for example PF-06835375. In some embodiments, the exogenous protein is a single chain antibody comprising the CDRs or heavy and light chain variable regions of these antibodies.

[0121] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form an antibody binding site. An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelised, CDR-grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled in soluble or bound form as well as fragments, including epitope-binding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences. An antibody may be from any species. The term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab', F(ab')2 single stranded antibody (svFC), dimeric variable region (Diabody) and disulphide-linked variable region (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Antibody fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. The skilled artisan will further appreciate that other fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH)~ Fc fusions and scFv-scFv-Fc fusions.

[0122] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

[0123] In some embodiments, engineering comprises introducing into the cell a sequence encoding the antibody, cytokine or chemokine. In some embodiments, introducing into a cell is integrating into the genome of the cell. In some embodiments, into the genome is into an endogenous antibody locus or endogenous antibody gene. In some embodiments, introducing a sequence is introducing a nucleic acid molecule comprising the sequence. In some embodiments, introducing a sequence comprises integrating the sequence into the genome of the cell. In some embodiments, engineering comprises introducing into the cell asequence encoding light chain of an exogenous antibody and a heavy chain of an exogenous antibody.

[0124] In some embodiments, engineering comprises introducing at least one first mutation into an endogenous heavy chain constant region. In some embodiments, the heavy chain of the exogenous antibody comprises at least one second mutation. In some embodiments, the first and second mutations induce heterodimerization. In some embodiments, the first and second mutations inhibit homodimerization. In some embodiments, inducing heterodimerization is inducing heterodimerization between the endogenous heavy chain and the exogenous heavy chain. In some embodiments, the engineering follows the scheme presented in Figure 8C. It will be understood that while Figure 8C shows a CD3 engager, the use of any exogenous protein is envisioned and encompassed in the scope of the invention.

[0125] Mutations that promote heavy chain heterodimerization and / or inhibit homodimerization are well known in the art. Any such mutations or alterations may be used for engineering the cell / antibodies of the invention. In some embodiments, the first mutation is selected from a mutation provided in Table 1 and the second mutation is provided in Table 1 and is a corresponding mutation to the first mutation. Examples of these mutations can be found in Table 1. In some embodiments, the mutation is selected from a mutation in Table 1. In some embodiments, the first mutation is selected from a group of mutation provided in a row and the second column of Table 1 and the second mutation is the group of mutations provided in that same row of Table 1 in the third column. The mutations in Table 1 are provided with the Kabat numbering for IgGl unless otherwise stated; corresponding mutations can be made in other IGs and specifically in other IgGs.

[0126] Table 1: Mutations for enhancing heterodimerization and inhibiting homodimerization of heavy chains.

[0127] In some embodiments, engineering comprises replacing the CHI domain of an endogenous heavy chain with a CL domain. In some embodiments, engineering comprises replacing the CL domain of an endogenous light chain with a CHI domain. This swapping of CHI and CL domains will ensure that the endogenous light chain pairs with the endogenous heavy chain. Since the introduced exogenous antibody has the CL and CHI domains in the correct positions the light chain from the exogenous antibody will not pair with the endogenous heavy chain and vice versa. In some embodiments, engineering comprises introducing an exogenous heavy chain of an antibody in which a CL domain hasreplaced the CHI domain. In some embodiments, engineering comprises introducing an exogenous light chain of an antibody in which a CHI domain has replaced the CL domain. Similar to the first swapping of CHI and CL domains, this will ensure that the exogenous light chain pairs with the exogenous heavy chain. Since the endogenous antibody has the CL and CHI domains in the correct positions the light chain from the exogenous antibody will not pair with the endogenous heavy chain and vice versa. In some embodiments, the engineering follows the scheme presented in Figure 8D. It will be understood that while Figure 8D shows a CD3 engager, the use of any exogenous protein is envisioned and encompassed in the scope of the invention.

[0128] In some embodiments, the method further comprises isolating the polyclonal antibody from the culture media. In some embodiments, all antibodies are isolated. In some embodiments, only engineered antibodies are isolated. In some embodiments, all antibodies are engineered and non-engineered antibodies. Methods of isolating antibodies are well known and include for example, affinity chromatography, ion exchange chromatography, size exclusion chromatography, precipitation, protein A / G / L beads, and IgG capture. Antibodies that bind to human IgGs and in particular IgGl are well known in the art and can be used to affinity purify the antibodies. Similarly, when an scFv or other exogenous antibody is added to engineered antibodies, these engineered antibodies can be affinity purified with antibodies against the exogenous molecule.

[0129] By another aspect, there is provided a method of enriching B cells, plasmablasts and / or plasma cells in culture, the method comprising receiving a population of immune cells and culturing the population in culture media for a time sufficient to enrich for B cells, plasmablasts and / or plasma cells in the culture.

[0130] In some embodiments, the B cells, plasmablasts and plasma cells are cancer derived B cells, plasmablasts and plasma cells. In some embodiments, cancer derived is tumor derived. In some embodiments, cancer derived is derived from a malignant effusion. In some embodiments, the population is a mixed population. In some embodiments, the population comprises B cells and T cells. In some embodiments, the population is depleted of T cells. In some embodiments, the population is depleted of CD3 positive cells. In some embodiments, the method further comprises depleting the population of T cells. In some embodiments, the method further comprises depleting the population of CD3 cells. In some embodiments, the depleting comprises isolating the T cells / CD3 cells and removing them from the population. In some embodiments, the culturing is for a time sufficient forexpansion of B cells. In some embodiments, the culturing is for a time sufficient for expansion of CD 19 positive cells. In some embodiments, the method further comprises after the expansion, isolating CD 19 positive cells from the culture. In some embodiments, the method further comprises further culturing the isolated CD 19 positive cells / B cells for a time sufficient to produce plasmablasts and / or plasma cells. In some embodiments, the time is sufficient to produce plasmablasts and plasma cells. In some embodiments, the time is sufficient to produce plasma cells. In some embodiments, the method is a method of enriching plasmablasts and / or plasma cells. In some embodiments, the method is a method of enriching cancer derived plasmablasts and / or plasma cells. In some embodiments, the method does not comprise a step of enriching for 4-1 BBL positive cells. In some embodiments, the method dose not comprise a step of isolating antibodies from immortalized cells. In some embodiments, the cells are primary cells. In some embodiments, the cells are not immortalized cells.

[0131] In some embodiments, the culturing is in the presence of cancer cells. In some embodiments, the culturing before isolating CD19 / B cells is in the presence of cancer cells. In some embodiments, the further culturing of the isolated CD19 / B cells is not in the presence of cancer cells. In some embodiments, not in the presence is in the absence of. In some embodiments, the media is media such as is described hereinabove. In some embodiments, the sufficient time is a time such as is described hereinabove.

[0132] In some embodiments, the method comprises loading the isolated polyclonal antibodies onto a population of T cells. In some embodiments, the loading produces a population of polyclonal antibody loaded T cells. In some embodiments, the T cells express an endogenous Fc receptor. Examples of Fc receptors include, but are not limited to FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIC (CD32C), FcyRIIIA (CD16A), FcyRIIIB (CD16B), FcsRI, FceRII (CD23), FcaRI (CD89), Fca / pR, FcpR (FAIM3 / TOSO), FcRn (FCGRT), and Fc8R. In some embodiments, the endogenous Fc receptor is an FcRgamma receptor. In some embodiments, the T cells express an exogenous Fc receptor. In some embodiments, the exogenous Fc receptor is an anti-Fc receptor chimeric antigen receptor (CAR) (e.g., an anti-FcR CAR). In some embodiments, the CAR is an anti-FcRg CAR. In some embodiments, the administering is administering the population of T cells loaded with the isolated polyclonal antibodies. In some embodiments, the CAR is selected form a CD16-CAR, a CD64-CAR, a CD32A-CAR, a CD89-CAR and a FcsRI-CAR.

[0133] In some embodiments, the method further comprises conjugating a drug to the isolated polyclonal antibodies. In some embodiments, the conjugating produces a polyclonal antibody-drug conjugate (ADC) composition. In some embodiments, the drug is a toxin. In some embodiments, the drug is cytotoxic. In some embodiments, the drug is lethal to a target cell. In some embodiments, the target cell is cancer. In some embodiments, the drug is a chemotherapeutic agent. In some embodiments, the drug is a poison. Drugs for producing ADCs are well-known in the art and any may be used. Examples of chemotherapeutics include, but are not limited to alkylating agents such as cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, and cisplatin; antimetabolites including methotrexate, 5-fluorouracil, capecitabine, cytarabine, gemcitabine, and fludarabine; topoisomerase inhibitors like doxorubicin, daunorubicin, idarubicin, etoposide, and topotecan; microtubuletargeting agents such as paclitaxel, docetaxel, vincristine, vinblastine, and vinorelbine; antitumor antibiotics including bleomycin, mitomycin C, actinomycin D, and mitoxantrone; and other agents like hydroxyurea, procarbazine, temozolomide, platinum compounds (carboplatin, oxaliplatin), and trabectedin. In some embodiments, conjugating is linking. In some embodiments, conjugating is covalently conjugating. In some embodiments, the administering is administering the ADC composition.

[0134] By another aspect, there is provided a population of B cells, plasmablasts and / or plasma cells produced by a method of the invention.

[0135] By another aspect, the is provided a composition comprising the population of the invention.

[0136] By another aspect, the is provided a composition comprising the antibodies, engineered antibodies and / or ADC compositions of the invention.

[0137] By another aspect, the is provided a composition comprising the loaded T cell population of the invention.

[0138] In some embodiments, the population of the invention is for use in treating cancer. In some embodiments, the composition of the invention is for use in treating cancer. In some embodiments, the population of the invention is for use in producing a medicament for treating cancer. In some embodiments, the composition of the invention is for use in producing a medicament for treating cancer.

[0139] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0140] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0141] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0142] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0143] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0144] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0145] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0146] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0147] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0148] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor LaboratoryPress, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Example 1:

[0149] It was hypothesized that polyclonal antibodies extracted from tumor infiltrating B lineage cells could be utilized to target tumors of the same type. These polyclonal antibodies were dubbed Oncologic Multi-Epitope General (OMG) Antibodies, and it was thought they can serve as an off-the-shelf therapy. Therefore, a single batch or multiple batches of OMG Antibodies may be utilized for one or multiple patients. An overview of the OMG therapy process is provided in Figure 1. Tumor infiltrating B lineage cells are collected from tumors or tumor-associated fluids, grown in culture conditions supporting B lineage cell proliferation and differentiation, antibodies secreted by the cultured cells are collected and purified and utilized as a polyclonal therapy to treat patients.

[0150] In order to optimize B cell culture conditions, peripheral blood mononuclear cells (PBMCs) from healthy individuals were collected (Fig. 2A). Enrichment for B cells and antibody secreting cells was performed by magnetic bead enrichment (Miltenyi #130-091-151) (Fig.2B). Cells were cultured in conditions supporting proliferation and differentiation (Immunocult Human B cell expansion media; STEMCell #100-0645). Viability (Fig. 2C), expansion rate (Fig. 2D) and phenotype of the cells (Fig. 2E) were characterized over time. The PBMC B-cells survived well cand could be continuously cultured and expanded with an increase in plasmablasts and antibody secreted cells and a decrease in migratory B cells and antigen presenting B cells over time. RPMI 1640 media supplemented with molecules for promoting B cell expansion was also tested (for example 10% FBS, 55 uM B-mercaptoethanol, HEPES 5 mM, R848 1 ug / ml, IL-2 10 ng / ml, IL- 10 10 ng / ml and IL-21 40 ng / ml) was found to produce comparable B cell and antibody expressing cell expansion. Thus, the culture conditions adopted are suitable for expanding tumor infiltrating B cells and producing OMG antibodies.

[0151] Tumor infiltrating B cells can be extracted from multiple tumor types (Fig. 1A and 3A). The frequency of B lineage cells was characterized over time and compared to PBMC cultures. Antibody production levels were also quantified. During late-stage culture, additional parameters such as B cell enrichment or cytokine supplementation were utilized for characterization.

[0152] Both T and B cells can be cultured from tumor fragments. As a control and comparison to B cell expansion, T cells were cultured from the same samples in base media supplemented with IL-2 (standard protocol or producing therapeutic TILs). B cells were cultured in B cell expansion media. While culture in IL-2 leads to T cell survival and modest expansion, culture of these cells in B cell expansion media leads to T cell death. CD 19+ B cells show a robust expansion in the B cell specific media (Fig. 3B-3C). Tumor samples from three cancer types, hepatocellular carcinoma (HCC), renal cell carcinoma (RCC) and pancreatic cancer, were used for extraction of immune cells. The percentage of CD 19 cells in the culture in B cell media was monitored overtime and it was observed that CD 19+ cells reached a peak between days 7-12 (Fig. 3D). The three cancers showed a similar peak time for CD 19+ B cell frequency (Fig. 3E). At later stages of culture, the CD 19+ cells decrease; likely due to differentiation into antibody secreting cells (CD19-).

[0153] The frequency of B cells in the culture was compared between a culture of all immune cells and one that had undergone B cell enrichment as proposed in the experimental method. Initially a mix of immune cells is cultured in the B cell expansion media. During the B cell peak, negative selection by magnetic beads is performed to produce a culture enriched for B cells. This produced an overall much greater B cell percentage in the enriched culture, reaching essentially 100% B cells when the starting culture was already depleted for T cells (CD3- culture) (Fig. 3F). The supplementation of IL-21 to the B cell culture media was also tested (Fig. 3G). IL-21 produced a beneficial proliferative boost and can be added to the culture to enhance cell number.Example 2: Testing OMG antibodies

[0154] Next the OMG antibodies were tested for their ability to bind tumor cell lines as well as primary tumor cells. First OMG antibodies derived from RCC tumors (isolated polyclonal antibodies derived from tumor infiltrating B cells (TIL-B) extracted from RCC tumors) were tested for binding to primary RCC tumor cells and robust binding was observed (Fig. 4 A, TIL-B Matched). Binding was significant as compared to binding to control cells. Further,binding increased in a dose dependent manner (Fig. 4D). The OMG antibodies from RCC tumors also bound to HCC tumor cell lines (HuH75, Hep3B and HepG2), although at lower levels of binding (Fig. 4A, TIL-B Unmatched). OMG antibodies derived from HCC tumors significantly bound HCC primary tumor cells and HCC cell lines (HCC RA374, HuH75, Hep3B and HepG2) (Fig. 4B, TIL-B Matched), just as OMG antibodies derived from pancreatic tumors bound primary pancreatic tumor cells and cell lines (Pane RA441R, RA-200p3p3, RA-454p3) (Fig. 4C, TIL-B Matched).

[0155] OMG antibodies were also compared to antibodies derived from plasma and antibodies derived from PBMCs of healthy individuals. OMG antibodies were superior to plasma antibodies from RCC patients (Fig. 4E), HCC patients (Fig. 4F) and pancreatic cancer patients (Fig. 4G). In the case of HCC OMG, the antibodies of the invention were significantly superior.

[0156] In order to quantify cancer cell killing, OMG antibodies derived from RCC tumors and healthy donor PBMC antibodies were added to a culture of primary RCC tumor cells with and without primary NK cells. Specific killing was measured and normalized to the culture without NK cells. OMG antibodies induced a dose dependent specific tumor cell killing, which was not observed with the PBMC antibodies (Fig.4H). This indicates that the OMG antibodies are effective for targeting and killing cancer cells. Importantly, the OMG antibodies did not substantially bind to cells matched control cells (T cells from PBMCs) (Fig. 41)Example 3: OMG antibodies from malignant effusions

[0157] Effusions and ascites from malignant cancers are known to be sources of tumor infiltrating immune cells. Tumor associated fluid samples, including pleural and ascites across multiple tumor types were assessed for lymphocyte infiltration and anti -tumor activity in co-culture experiments. First, the cells from pleural effusions and ascites from 7 different types of cancers were assessed. Antibody expressing cells (BD19+ / BCMA-, CD19+ / BCMA+ and CD19- / BCMA+ cells) were present in the fluids from all cancers tested (Fig. 5A). Plasmablasts and antibody secreting cells were found in as high or even higher concentrations as B cells (Fig. 5B). Further, malignant effusion antibody producing cells strongly skewed toward an activated phenotype (BMCA+ / CD19+ or IgG+ / IgM+) (Fig.5C).

[0158] The total IgG per sample from ascites was in the range of 10A3-10A4 mg per sample, indicating that malignant effusions are a rich source of anti-tumor antibodies even withoutculturing the antibody expressing cells. As a proof of concept, antibodies were collected from uterine cervix adenocarcinoma ascites and the plasma cells from this ascites were also cultured as before, after which polyclonal antibodies were collected from the culture. Both the ascites antibodies and the plasma cell (PC) culture antibodies produced dose dependent specific killing of cancer cells from the ascites fluid in the presence of NK cells (Fig. 5D).Antibodies from PBMCs did not produce specific killing. Further, OMG antibodies were purified from ascites or pleural effusions from pancreatic, hepatic, brain, renal, bladder, head and neck, ovarian and breast cancers and all were found to bind cell lines and patient-derived tumor cells of the same cell type (Fig. 5E). Thus, B cells, plasmablast and plasma cells from malignant effusions are viable sources for OMG antibodies (Fig. 6A-6B) and the antibodies in the effusions themselves can also be used.

[0159] The polyisotypic antibody repertoire from ascitic fluid or produced by culture of B cells from ascites (in immunocult) was compared. Ascites from a gastrointestinal tumor was collected and antibodies and B cells were extracted from the fluid. The B cells were cultured and the supernatant was evaluated. IgG, IgM and IGA antibodies (other isotypes were not evaluated) were found both in the ascites and in the supernatant from the B cells (Fig. 6C).The amount of each isotype was similar between the two sources, though the ascites cells showed more IgA and fewer IgM antibodies.Example 4: Pooled OMG antibodies

[0160] One of the major advantages of OMG antibodies is their polyclonality. Current anticancer antibody therapeutics rely on monoclonal antibodies. This can entail screening of tumor reactive antibodies, characterization of their targets and selecting a single antibody to expand thus producing a therapeutic monoclonal antibody (see for example Biswas et al. "IgA transcytosis and antigen recognition govern ovarian cancer immunity", Nature 591.7850 (2021): 464-470). However, monoclonal antibodies are stymied by tumor heterogeneity. If not all cells of the tumor express the target antigen, the monoclonal therapeutic will leave residual disease which will cause relapse and new tumors that are refractory to the targeted therapy. The polyclonality of OMG antibodies does not suffer from this defect and it will include antibodies against all the cells of the cancer.

[0161] To demonstrate the superiority of a vast repertoire of polyclonal antibodies, polyclonal OMG antibodies from a single donor (antibodies from ascites) were compared with pooled OMG antibodies from multiple donors of the same tumor type (ascites and Bcell cultures from tumor and ascites) for their ability to bind cancer cells of that tumor type but from other patients. For all four cancers tested (renal-Fig. 7A; head and neck-Fig. 7B; breast-Fig. 7C; ovarian-Fig. 7D) the pooled antibodies were always significantly superior to the antibodies from only one donor. A killing assay was also performed with breast tumor cells isolated from an additional patient in presence of primary NK cells. While polyclonal OMG cells from a single breast cancer patient were able to produce increased specific killing, the pooled antibodies from multiple donors were significantly better (Fig. 7E). This demonstrates the superiority of using polyclonal antibodies with their broad repertoire of binding targets.Example 5: Oncologic Multi-epitope Engineered General Antibody (OMEGA)

[0162] In order to improve the antibodies’ therapeutic potential and overall yield, the antibody secreting cells were engineered to produce polyclonal engineered antibodies termed Oncologic Multi-epitope Engineered General Antibodies (OMEGA). The approach is similar to the OMG antibody process with an additional gene editing step of the polyclonal tumor derived TIL-B cultures. During this process, engineered cultures may or may not be enriched for engineering. Similarly, the purified antibodies resulting from this process may or may not be enriched for the engineered antibody fraction. The starting material may be tumor resections or effusions.

[0163] Exemplary universal polyclonal engineering strategies are depicted in Figures 8A-8D. Either the light chain loci or the heavy chain constant segments were engineered. This enables fusion of the antibody with functional domains. An example of a functional domain may be a T cell engager domain enabling recruitment of T effector cells to the tumor. Targeting the constant domains enables the engineering strategy to retain the original polyclonal anti-tumor specificity. For Figures 8A-8D, the target locus and transgene integration site are indicated above, and the resulting antibody is depicted below. For Figures 8A-8D, the specificity of the polyclonal antibodies is not affected, as indicated by the colorful scheme of the variable segments of the antibodies. An anti-CD3 domain is employed as an example for a fusion protein enabling supraphy si ologi cal activity, though any modification is envisioned.

[0164] Figure 8A shows an engineering strategy to fuse polyclonal OMG antibodies with a payload at the heavy chain C-terminus of the antibodies. The transgene integrates in the last exon of a secreted heavy chain constant segment. Figure 8B shows an engineering strategyto fuse polyclonal OMG antibodies at the light chain C-terminus of the antibodies. This strategy enables that all isotypes secreted by the TIL-B cultures are targeted for engineering. This strategy may enable antibody multimerization commonly occurring with IgA and IgM isoforms. Either or both IgK and IgL light chain constants may be targeted. Figure 8C shows an engineering strategy to generate mispairing free, bi-specific antibodies. Early exons in a specific IgH isotype are targeted for transgene integration. The transgene codes for the rest of the targeted heavy chain mutated to include a Knob, for a full light chain with an anti-CD3 VH and for an anti-CD3 VL fused to an heavy chain constant domain including a Hole. The Knob-in-Hole mutations enable heavy chain dimerization with minimal mispairing. The VH-CL and VL-CH fusions enable light chain pairing with minimal mispairing. The Knob and Hole mutation locations may be swapped and any other mutation pairs for producing heterodimerization (see Table 1) may be employed. Figure 8D shows an engineering strategy to produce polyclonal bi-specific T cell engagers (BiTE) or similar molecules devoid of full heavy chain constant segments. Here, the payload is integrated in the VH-CH intron of the IgH locus. The transgene includes a full CHI domain to enable pairing with the endogenous light chain and is fused with a linked to an anti-CD3 scFv domain. This enables secretion of low half-life bi-specific polyclonal T cell engagers that can reach solid tumors more conveniently than full antibodies.

[0165] Polyclonal antibody expressing cells were engineered as depicted in Figure 8B using CRISPR-Cas9 mediated cleavage of the IgK constant segment. The anti-CD3 scFv clone OKT3 was inserted. Cleavage was monitored using TIDE analysis (Fig. 9A). Significant rates of engineering approaching 15% of cells were observed as determined by measuring His-tag incorporation (Fig. 9B). Only cells expressing the IgK light chain are engineered, as expected from the engineering strategy employed (Fig. 9C).

[0166] Supernatants were taken and antibody binding to T cells was evaluated by flow cytometry (Fig. 9D). The engineered antibodies derived from the supernatant bound T cells at a significantly higher level than non-engineered cells (Fig. 9E). Following enrichment of engineered B cells using magnetic beads that bind the His Tag, the supernatant from only engineered cells was also tested. This enriched supernatant was by far superior to unengineered cells or a mix (no enrichment performed) of engineered and un-engineered cells (Fig. 9F)

[0167] This engineering strategy was also applied to B cells from ascites from a patient with a gastrointestinal (GI) tumor, pleural effusion from a patient with breast cancer and a hepatictumor (HCC). Cells were cultured for 7-14 days, electroporated with CRISPR-Cas9 RNP and transduced with AAV-6 AAVs containing the donor cassette. Engineering was successful in all of the cells tested and at levels comparable to what was achieved in PBMCs (Fig. 10A). Engineered antibodies from the GI patient’s cells were tested for binding to PBMC derived primary human T cells (CD3 positive) and T cells binding by the CD3 engager was observed (Fig. 10B). To confirm that polyclonality against the desired tumor was retained the OMEGA antibodies from the HCC tumor were used to stain both the HuH7.5 tumor cell line and the HCC281024 patient-derived tumor cell line (Fig. IOC).Polyclonal binding was clearly retained, indicating that the antibody engineering had not had a deleterious effect.

[0168] Similar results were obtained with a second anti-CD3 scFv: TR66. In fact, this T cell engager was found to produce better engineering rates (Fig. 11 A) and increased T cell binding (Fig. 11B). This supports the universality of the engineering strategy and indicates it is not limited to one specific insertion.

[0169] Polyclonal antibody expressing cells were also engineered as depicted in Figures 8C and 8D. The knob-in-holes (KiH) approach produced nearly 50% engineered cells (Fig.12A) with good T cell binding (Fig. 12B) and tumor specificity (Fig. 12C). While BiTE production was also highly effective, although at slightly reduced levels as compared to the KiH approach (Fig. 12D-F).Example 6: OMG antibodies outperform monoclonal therapeutics

[0170] Pools for OMG antibodies from multiple donors with the same malignancy type were compared to cutting edge monoclonal antibody therapy. The anti-EGFR antibody Cetuximab is currently a first line therapy for both head and neck cancer and lung cancer, anti-GPC3 antibodies have been proposed for treating hepatic cancer and anti-HER2 and anti-HER3 antibodies are used for treating breast cancer. Flow cytometry was used to evaluate tumor cell line binding for OMG antibodies (from head and neck, lung or hepatic cancers), for cetuximab (R&D Biosystems FAB9577P), for an anti-GPC3 antibody (NovusBio NBP2-44486), for an anti-Her2 antibody (Miltenyi 130-124-475) and an anti-Her3 antibody (Miltenyi 130-107-901). As can be seen in Figure 13, the OMG antibodies demonstrate an improved breadth of binding as compared to the monoclonal antibodies tested.Example 7: Additional OMG and OMEGA antibody configurations

[0171] OMG antibodies are also loaded onto FcR-based cell therapies (native or engineered NK cells or T cells) in a manner similar to the loading of monoclonal antibodies and used as a polyclonal therapeutic (see for example, Constantinides et al., “The arming of natural killer cells with Fc-engineered monoclonal antibodies confers specificity against tumor B cells”, MedComm, 2020, Jul 4;6(7):e70242, and Caratelli et al., “FCgamma chimeric receptor-engineered T cells: methodology, advantages, limitations, and clinical relevance”, Front. Immunol., 2017, Apr. 27:8:457, the contents of which are hereby incorporated by reference in its entirety). In particular FcR CAR-T cells are precultured with polyclonal OMG antibodies to produce a polyclonal CAR-T population. This population is broadly effective against tumors from the same type of cancers from which the OMG antibodies were derived. Killing of cancer cells is confirmed by coculture of the OMG-CAR-T cells and the target cancer cells. FcR CAR-T cells not pre-incubated with OMG antibodies are used as a negative control.

[0172] Antibody drug conjugates (ADCs) are also generated with the OMG antibodies and OMEGA antibodies. Toxins, chemotherapeutics or other killing moi eties are conjugated to the OMG or OMEGA antibodies by methods known in the art (e.g., conjugated to the C-terminus of the heavy chain, or site-specific conjugation to the Fc region). The enhanced killing of the ADCs is confirmed in culture with target cancer cells of the same type as the cancer from which the antibodies were derived.

[0173] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. A method of treating cancer in a subject in need thereof, the method comprising:a. receiving plasmablasts and / or plasma cells from a malignant effusion from a subject suffering from cancer that is not the subject in need of treatment;b. culturing said received plasmablasts or plasma cells in culture media;c. isolating a composition of polyclonal antibodies from said culture media; andd. administering said composition of polyclonal antibodies to said subject in need of treatment;thereby treating cancer in a subject in need thereof.

2. The method of claim 1, wherein said cancer in said subject in need of treatment is the same type of cancer as said cancer that produced the malignant effusion.

3. The method of claim 1, wherein said cancer in said subject in need of treatment is a different type of cancer as said cancer that produced the malignant effusion.

4. The method of any one of claims 1 to 3, wherein said cancer is selected from renal cancer, liver cancer, pancreatic cancer, lung cancer, gastric / gastrointestinal cancer, ovarian cancer, breast cancer, skin cancer, head and neck cancer, uterine cancer, brain cancer, bladder cancer, and cervical cancer.

5. The method of claim 4, wherein said renal cancer is renal cell carcinoma (RCC), or said liver cancer is hepatocellular carcinoma (HCC).

6. The method of any one of claims 1 to 5, wherein said receiving comprises receiving a malignant effusion sample, isolating CD 19 positive cells from said sample and culturing said isolated CD 19 cells in media to enrich for plasmablasts and plasma cells.

7. The method of any one of claims 1 to 5, wherein said receiving comprises receiving a malignant effusion sample, and isolating cells positive for syndecan 1 (CD138 / SDC1), B-cell maturation antigen (BCMA / TBFRSF17), CD38 or a combination thereof from said sample, thereby isolated plasmablasts and / or plasma cells.

8. The method of any one of claims 1 to 5, wherein said receiving comprises receiving a malignant effusion sample, isolating immune cells from said sample, culturing said isolated immune cells in B cell media for a time sufficient to expand a CD 19+ B cell population, isolating CD 19 positive cells from said culture and further culturing said isolated CD 19 positive cells to produce plasmablasts and plasma cells.

9. The method of any one of claims 1 to 8, wherein said method does not comprise a step of enriching for 4-1BBL positive cells or isolating antibodies from immortalized cells.

10. The method of any one of claims 1 to 9, wherein said malignant effusion is selected from an ascites and a pleural effusion.

11. The method of any one of claims 1 to 10, wherein said media is B cell culture media.

12. The method of any one of claims 6 to 11, wherein said enriching comprises culturing for an amount of time sufficient to produce a culture comprising at least 30% plasmablasts and plasma cells.

13. The method of any one of claims 1 to 12, comprising performing steps a-c with plasmablasts and / or plasma cells from a malignant effusion from a plurality of subjects suffering from cancer, wherein said plurality comprises a first subject suffering from cancer and a second subject suffering from cancer, wherein said first and second subject are different subjects, and wherein said administering comprising administering polyclonal antibody isolated from said received plasmablasts and / or plasma cells from said plurality of subjects.

14. The method of claim 13, wherein said plurality of subjects all suffer from the same type of cancer as said subject in need of treatment.

15. The method of any one of claims 1 to 14, further comprising genetically engineering said B cells, plasmablasts or plasma cells during said culturing, wherein said genetic engineering comprises linking an exogenous protein to an endogenous heavy chain or light chain constant region or fragment thereof.

16. The method of claim 15, wherein said genetic engineering comprises RNA-guided nuclease engineering, optionally wherein said RNA-guided nuclease engineering is CRISPR-Cas engineering.

17. The method of claim 15 or 16, wherein said exogenous protein is selected from a T cell engager, a cytokine or chemokine, and anti-immune protein antibody or antigen binding fragment thereof.

18. The method of claim 17, wherein said T cell engager is an anti-CD3 single chain antibody or anti-CD3 single domain antibody.

19. The method of claim 18, wherein said T cell engager is an anti-CD3 single chain antibody and comprises an amino acid sequence selected from SEQ ID NO: 1-3.

20. The method of claim 17, wherein said cytokine or chemokine is selected from interleukin 15 (IL-15), IL-7, IL-2, IL-21, IL-12, IL-4, C-X-C motif chemokine 12 (CXCL12), and CXCL13.

21. The method of claim 17, wherein said anti -immune protein antibody is an antibody against an immune protein selected from CD28, CD40, Programmed death-ligand 1 (PD-L1), Cytotoxic T-lymphocyte associated protein 4 (CTLA4), C-X-C chemokine receptor type 4 (CXCR4), and CXCR5.

22. The method of any one of claims 15 to 19, wherein said genetic engineering comprises introducing at least one first mutation into an endogenous heavy chain constant region, introducing a sequence encoding an anti-CD3 antibody’s light chain, introducing a sequence encoding said anti-CD3 antibody’s heavy chain and wherein said anti-CD3 antibody’s heavy chain comprises at least one second mutation and wherein said first and second mutations induce heterodimerization between said endogenous heavy chain constant region and said anti-CD3 antibody’s heavy chain and inhibit homodimerization.

23. The method of claim 21, wherein said genetic engineering comprises introducing at least one first mutation into an endogenous heavy chain constant region, introducing a sequence encoding an anti-immune protein antibody’s light chain, introducing a sequence encoding said anti-immune protein antibody’s heavy chain and wherein said anti -immune protein antibody’s heavy chain comprises at least one second mutation and wherein said first and second mutations induce heterodimerization between said endogenous heavy chain constant region and said anti -immune protein antibody’s heavy chain and inhibit homodimerization.

24. The method of claim 22 or 23, wherein said first and second mutations are selected from Table 1.

25. The method of any one of claims 15 to 19, wherein said genetic engineering comprises introducing a sequence encoding an anti-CD3 antibody’s light chain and introducing a sequence encoding said anti-CD3 antibody’s heavy chain, whereina. a CL domain of said anti-CD3 antibody’s light chain has been replaced with a CHI domain and a CHI domain of said anti-CD3 antibody’s heavy chain has been replaced with a CL domain; orb. a CL domain of an endogenous light chain has been replaced with a CHI domain and a CHI domain of an endogenous heavy chain has been replaced with a CL domain.

26. The method of claim 21, wherein said genetic engineering comprises introducing a sequence encoding an anti -immune protein antibody’s light chain and introducing a sequence encoding said anti -immune protein antibody’s heavy chain, whereina. a CL domain of said anti-immune protein antibody’s light chain has been replaced with a CHI domain and a CHI domain of said anti-immune protein antibody’s heavy chain has been replaced with a CL domain; orb. a CL domain of an endogenous light chain has been replaced with a CHI domain and a CHI domain of an endogenous heavy chain has been replaced with a CL domain.

27. The method of any one of claims 1 to 26, wherein said composition is a pharmaceutical composition comprising said polyclonal antibodies and a pharmaceutically acceptable carrier, excipient or adjuvant.

28. The method of claim 27, wherein said pharmaceutical composition consists of said polyclonal antibodies and a pharmaceutically acceptable carrier, excipient or adjuvant.

29. The method of any one of claims 1 to 27, comprising loading said isolated polyclonal antibodies onto a population of T cells expressing an anti-Fc receptor chimeric antigen receptor (CAR) to produce a population of polyclonal antibody loaded T cells andwherein said administering is administering said population of polyclonal antibody loaded T cells.

30. The method of any one of claims 1 to 27, comprising conjugating a drug to said isolated polyclonal antibodies to produce a polyclonal antibody-drug conjugate (ADC) composition and wherein said administering is administering said ADC composition.

31. A pharmaceutical composition comprising polyclonal antibodies harvested from B cells, plasmablasts and / or plasma cells derived from a malignant effusion in culture and a pharmaceutically acceptable carrier, excipient or adjuvant.

32. The pharmaceutical composition of claim 31, for use in treating cancer.

33. A method of enriching tumor derived B cells, plasmablasts and / or plasma cells in culture, the method comprising receiving a mixed population of primary immune cells from a tumor, culturing said mixed population in culture media for a time sufficient to enrich B cells, plasmablasts and / or plasma cells in the culture, wherein said culture is selected from:a. Immunocult Human B cell expansion media;b. Immunocult Human B cell expansion kit supplemented with interleukin 21 (IL- 21);c. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, RP105, and IL-7;d. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, RP105, and IL-4;e. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, RP105, IL-4, and IL-21;f. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, megaCD40L, CpG ODN2006, IL-10, IL-2, IL-15, and IL-6;g. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, megaCD40L, IL- 4, IL-21, and B-cell activating factor (BAFF / TNFSF13B);h. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, megaCD40L, and 1 IL-4; andi. RPMI 1640 supplemented with FBS, beta-mercaptoethanol, HEPES, R848, IL-2, IL- 10 and IL-21.

34. The method of claim 33, wherein said culturing comprises culturing for time sufficient for expansion of a CD19 positive B cell population, isolating CD19 positive cells from said culture and further culturing said isolated CD 19 positive cells to produce plasmablasts and plasma cells and wherein the method is a method of enriching tumor derived plasmablasts and / or plasma cells.

35. The method of claim 33 or 34, wherein said mixed population is depleted of CD3 positive cells.

36. The method of any one of claims 33 to 35, wherein said method does not comprise a step of enriching for 4-1BBL positive cells or isolating antibodies from immortalized cells.

37. A population of tumor derived B cells, plasmablasts and / or plasma cells produced by a method of any one of claims 33 to 36.

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