Tumor-specific Cell Surface Antigens Research Articles

A combined immunopeptidomics, proteomics, and cell surface proteomics approach to identify immunotherapy targets for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG), recently reclassified as a subtype of diffuse midline glioma, is a highly aggressive brainstem tumor affecting children and young adults, with no cure and a median survival of only 9 months. Conventional treatments are ineffective, highlighting the need for alternative therapeutic strategies such as cellular immunotherapy. However, identifying unique and tumor-specific cell surface antigens to target with chimeric antigen receptor (CAR) or T-cell receptor (TCR) therapies is challenging. In this study, a multi-omics approach was used to interrogate patient-derived DIPG cell lines and to identify potential targets for immunotherapy. Through immunopeptidomics, a range of targetable peptide antigens from cancer testis and tumor-associated antigens as well as peptides derived from human endogenous retroviral elements were identified. Proteomics analysis also revealed upregulation of potential drug targets and cell surface proteins such as Cluster of differentiation 27 (CD276) B7 homolog 3 protein (B7H3), Interleukin 13 alpha receptor 2 (IL-13Rα2), Human Epidermal Growth Factor Receptor 3 (HER2), Ephrin Type-A Receptor 2 (EphA2), and Ephrin Type-A Receptor 3 (EphA3). The results of this study provide a valuable resource for the scientific community to accelerate immunotherapeutic approaches for DIPG. Identifying potential targets for CAR and TCR therapies could open up new avenues for treating this devastating disease.

Abstract 1780: TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements

Abstract The clinical success of cancer immunotherapy, including engineered T cell therapies, has revolutionized treatment paradigms and patient outcomes. While hematological tumors have benefited most from cell therapy approaches, the treatment of solid tumors remains a challenge in part due to the limited availability of abundant and tumor-specific cell-surface antigens. Peptides derived from intracellular tumor-specific proteins, such as cancer-testis antigens (CTAs), that are presented via HLA (pHLA) enable a therapeutic opportunity to target tumors while sparing normal tissue. Surface-accessible pHLA complexes may be targeted with engineered T cell receptors or TCR mimetic antibodies reformatted to chimeric antigen receptors (CARs). Using MAGE-A4 as a model CTA, we compared engineered human TCR- and CAR-T cells head-to-head to understand how to best deploy these modalities. To this end, we generated fully-human, HLA-A2/MAGE-A4(230-239)-specific TCR and CARs harboring CD28/CD3z or 41BB/CD3z signaling domains. TCR and CAR-T cells demonstrated similar robust on-target reactivity, cytokine release, and target cell lysis in vitro. In vivo, each of these modalities showed potent, dose-dependent anti-tumor efficacy against human xenograft tumors expressing low, endogenous levels (~500 cell-surface copies) of the MAGE-A4 peptide. However, differences emerged when we examined the in vivo kinetics and durability of tumor suppression. MAGE-A4 CD28/z CAR-T demonstrated the most rapid and potent tumor clearance, while the 41BB/z CAR-T showed delayed but ultimately complete efficacy. TCR-T cells induced tumor regressions during the first 2 weeks of treatment, but this response was transient and followed by tumor relapse. These differential responses correlated with early, modest accumulation of CD28/z CAR-T in line with fast tumor clearance. 41BB/z CAR-T showed a remarkable ~800-fold expansion in the tumor versus limited in vivo TCR-T proliferation. Mechanistically, the MAGE-A4 TCR induced strong CD3 proximal signaling associated with a greater induction of T cell dysfunction markers and limited cytotoxic potential in vitro. However, stimulating 41BB signaling pathways in the MAGE-A4 TCR T cells augmented long-term cytotoxicity. These data demonstrate that tumor-specific pHLA complexes can be potently targeted by both TCR and CAR-T cells, and that co-stimulatory signaling is necessary to mediate durable anti-tumor activity. Citation Format: Corinne E. Decker, Jacqueline Idun, Katja Mohrs, Thomas Meagher, Kevin Bray, Iryna Petriv, Jonathon Golas, Timothy Helms, Dharani Ajithdoss, Gavin Thurston, John Lin, Jessica R. Kirshner, David J. DiLillo. TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1780.

Single-cell transcriptome sequencing reveals potential novel combination of biomarkers for antibody-based cancer therapeutics in hepatocellular carcinoma.

Background: Antibody-based cancer therapeutics is developing rapidly in recent years for its advantages in precisely targeting the tumor cells. However, tumor-specific cell surface antigens are still lacking, and the heterogeneity of tumor mass greatly impeded the development of effective drugs. Methods: In the present study, single-cell RNA sequencing was used to dissect tumor heterogeneity in human hepatocellular carcinoma (HCC). Tissues from different spatial regions including the tumor, para-tumor, and distant normal liver tissues were dissociated into single cells, and the gene expressions were compared in a different subpopulation of cells from these regions and validated in independent cohorts. Results: A total of 28 cell clusters with different distribution patterns and gene expression profiles were identified within a heterogenous tumor and its paired liver tissues. Differentially expressed genes encoding the plasma membrane in cells with hepatic lineage were further extracted from single-cell transcriptome sequencing and validated in TCGA database. A 3-gene signature was identified to be significantly upregulated in dominant HCC tumor cell subpopulations with prognostic significance and validated in multiple independent patient cohorts. Conclusion: The composition of the three plasma membrane proteins on the surface of HCC tumor cells within a heterogenous tumor might indicate poor prognostic tumor subpopulations during cancer evolution and potential therapeutic targets.

Chimeric Antigen Receptor T Cells Targeting Cell Surface GRP78 to Eradicate Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a serious, life-threatening hematological malignancy. The treatment outcome of relapsed or refractory AML patients remains dismal, and new treatment options are needed. Chimeric antigen receptor (CAR) T cells have been successful in improving the prognosis for B-lineage acute lymphoblastic leukemia and lymphoma by targeting CD19. However, CAR T-cell therapy for AML is still elusive, owing to the lack of a tumor-specific cell surface antigen and spare hematopoietic stem cells (HSCs). This study generated a novel CAR construction that targets the cell surface protein glucose-regulated protein 78 (GRP78) (csGRP78). We confirmed that GRP78-CAR T cells demonstrate an anti-tumor effect against human AML cells in vitro. In xenograft models, GRP78-CAR T cells effectively eliminate AML cells and protect mice against systemic leukemia, in the meanwhile, prolonging survival. In addition, GRP78-CAR T cells also specifically eradicate the primary AML patient-derived blast. In particular, GRP78-CAR T cells spare normal HSCs, highlighting that GRP78-CAR is a promising approach for the therapy of AML.

T-cell-engaging Therapy for Solid Tumors.

T-cell engagers (TCE) are a rapidly evolving novel group of treatments that have in common the concurrent engagement of a T-cell surface molecule and a tumoral cell antigen. Bispecific antibodies and genetically engineered adoptive cell therapies, as chimeric antigen receptors or T-cell receptors, have similarities and differences among their mechanisms of action, toxicity profiles, and resistance pathways. Nevertheless, the success observed in the hematologic field has not been obtained with solid tumors yet, as they are biologically more complex and have few truly tumor-specific cell surface antigens that can be targeted with high avidity T cells. Different strategies are under study to improve their short-term perspective, such as new generations of more active TCEs, multi-target or combination of different treatments approaches, or to improve the manufacturing processes. A comprehensive review of TCEs as a grouped treatment class, their current status, and research directions in their application to solid tumors therapeutics are discussed here.

ALPPL2 Is a Highly Specific and Targetable Tumor Cell Surface Antigen.

Identification of tumor-specific cell surface antigens has proven challenging, as the vast majority of tumor-associated antigens are also expressed in normal tissues. In mesothelioma, we identified a highly specific tumor cell surface antigen that can be targeted for therapy development. Mesothelioma is caused by malignant transformation of the mesothelium, is incurable, and can be categorized into three histologic subtypes: epithelioid, biphasic, and sarcomatoid. To identity novel mesothelioma cell surface antigens with broad subtype coverage and high tissue specificity, we have previously selected phage antibody display libraries on live mesothelioma cells and tissues following counterselection on normal cells and identified a panel of human antibodies that bind all subtypes of mesothelioma, but not normal mesothelium. One of the antibodies, M25, showed high specificity against an antigen we identify here as ALPPL2. IHC on normal human tissues found that ALPPL2 is expressed only on placental trophoblasts, but not on any other normal tissues. This significant tissue specificity and broad tumor type coverage suggest that ALPPL2 could be an excellent cell surface target for therapeutic development against mesothelioma. To evaluate therapeutic potential of ALPPL2 targeting, an ALPPL2-targeted antibody-drug conjugate was developed and demonstrated potent and specific tumor killing in vitro and in vivo against both epithelioid and sarcomatoid mesothelioma. Thus, ALPPL2 belongs to a rare class of cell surface antigens classified as truly tumor specific and is well suited for therapy development against ALPPL2-expressing tumors. SIGNIFICANCE: These findings identify ALPP2 as a true tumor-specific cell surface antigen whose tissue specificity enables the development of novel therapies.

Lysosomal P-gp-MDR1 Confers Drug Resistance of Brentuximab Vedotin and Its Cytotoxic Payload Monomethyl Auristatin E in Tumor Cells.

Antibody-drug conjugates (ADCs) are composed of an antibody linked to cytotoxic anticancer payloads. ADCs recognize tumor-specific cell surface antigens and are internalized into lysosomes where proteolytic enzymes release the cytotoxic payloads. Efflux transporters on plasma membrane that play a significant role on multi-drug resistance in chemotherapy can be internalized on lysosomal membrane and sequester the cytotoxic payloads. In the present study, ATP binding cassette (ABC) efflux transporters including breast cancer resistance protein (BCRP), P-glycoprotein (P-gp-MDR1), multidrug resistance protein (MRP) 2, MRP3 and MRP4 in lysosomal, and plasma membrane of tumor cells were quantified by targeted quantitative proteomics. The cytotoxicity of brentuximab vedotin and its cytotoxic payload monomethyl auristatin E (MMAE) to the tumor cell lines in the presence and absence of elacridar (P-gp-MDR1 inhibitor) or chloroquine (lysosomotropic agent) were evaluated. MMAE is a substrate for P-gp-MDR1, as the apparent efflux ratio in MDR1 transfected MDCK cell monolayers was 44.5, and elacridar abolished the MMAE efflux. Cell lines that highly express P-gp-MDR1 show higher EC50s toward the cell killing effects of MMAE. Co-incubation with chloroquine or elacridar resulted in left shift of MMAE EC50 by 2.9–16-fold and 4.2–22-fold, respectively. Similarly co-incubation with chloroquine or elacridar or in combination of chloroquine and elacridar increased cytotoxic effects of brentuximab vedotin by 2.8- to 21.4-fold on KM-H2 cells that express a specific tumor antigen CD30 and P-gp-MDR1. These findings demonstrate important roles of P-gp-MDR1 on cytotoxic effects of brentuximab vedotin and its payload MMAE. Collectively, ABC transporter-mediated drug extrusion and/or sequestration needs to be early assessed for selection of optimal payloads and linkers when developing ADCs.

EGFRvIII Is Expressed in Cellular Areas of Tumor in a Subset of Glioblastoma.

Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific cell surface antigen often expressed in glioblastoma and has drawn much attention as a possible therapeutic target. We performed immunohistochemistry on histology sections of surgical specimens taken from 67 cases with glioblastoma, isocitrate dehydrogenase-wild type, and evaluated the morphological characteristics and distribution of the EGFRvIII-positive tumor cells. We then evaluated the localization of EGFRvIII-expression within the tumor and peritumoral areas. EGFRvIII immunopositivity was detected in 15 specimens taken from 13 patients, including two recurrent specimens taken from the same patient at relapse. Immunofluorescence staining demonstrated that EGFRvIII-positive cells were present in cells positive for glial fibrillary acidic protein (GFAP), and some showed astrocytic differentiation with multiple fine processes and others did not shown. The EGFRvIII-positive cells were located in cellular areas of the tumor, but not in the invading zone. In the two recurrent cases, EGFRvIII-positive cells were markedly decreased in one case and retained in the other. With regard to overall survival, univariate analysis indicated that EGFRvIII-expression in patients with glioblastoma was not significantly associated with a favorable outcome. Double-labeling immunofluorescence staining of EGFRvIII and GFAP showed that processes of large, well differentiated, GFAP-positive glia extend to and surround less differentiated, EGFRvIII-positive glial cells in cellular areas of tumor. However, in the tumor periphery, EGFRvIII-positive tumor cells were not observed. This finding suggests that EGFRvIII is involved in tumor proliferation, but that invading glioma cells lose their EGFRvIII expression.

Therapeutic Antibodies against Intracellular Tumor Antigens.

Monoclonal antibodies are among the most clinically effective drugs used to treat cancer. However, their target repertoire is limited as there are relatively few tumor-specific or tumor-associated cell surface or soluble antigens. Intracellular molecules represent nearly half of the human proteome and provide an untapped reservoir of potential therapeutic targets. Antibodies have been developed to target externalized antigens, have also been engineered to enter into cells or may be expressed intracellularly with the aim of binding intracellular antigens. Furthermore, intracellular proteins can be degraded by the proteasome into short, commonly 8–10 amino acid long, peptides that are presented on the cell surface in the context of major histocompatibility complex class I (MHC-I) molecules. These tumor-associated peptide–MHC-I complexes can then be targeted by antibodies known as T-cell receptor mimic (TCRm) or T-cell receptor (TCR)-like antibodies, which recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells. Advances in the production of TCRm antibodies have enabled the generation of multiple TCRm antibodies, which have been tested in vitro and in vivo, expanding our understanding of their mechanisms of action and the importance of target epitope selection and expression. This review will summarize multiple approaches to targeting intracellular antigens with therapeutic antibodies, in particular describing the production and characterization of TCRm antibodies, the factors influencing their target identification, their advantages and disadvantages in the context of TCR therapies, and the potential to advance TCRm-based therapies into the clinic.

Abstract 5332: Evaluation of GD2 expression in patients with osteosarcoma

Abstract GD2 is a disialoganglioside that is expressed on the cell surface in specific normal and malignant human tissues. Tumor specific cell surface antigens serve as important targets in cancer therapy. Anti-GD2 therapy using antibodies targeted to the cell surface antigen GD2 to induce antibody-dependent cellular cytotoxicity has been proven effective in treating GD2 expressing tumors like neuroblastoma and melanoma. Prior studies of osteosarcoma have shown GD2 to be expressed in malignant cells of osteosarcoma patients. In this study, the expression of GD2 in osteosarcoma will be evaluated further. A previously created and described tissue microarray was stained with 14.G2a, a murine monoclonal anti-GD2 antibody that was obtained from the National Cancer Institute. The intensity and location of tissue staining were assessed by a comparison between the positive and negative control slides. To evaluate GD2 expression quantitatively a cell-based ELISA was performed on different osteosarcoma patient derived cell cultures. The results of the tissue microarray showed that 34.8% of patient samples stained positive for GD2 while 60.9% of samples showed intermediate staining and only 4.3% of samples showed negative staining for GD2 (n=88). Nearly all of the osteosarcoma cell lines (n=24) showed levels of GD2 expression comparable to the neuroblastoma and fibrosarcoma cell lines and in some cases GD2 expression was significantly greater. Anti-GD2 therapy has been proven effective in treating patients with neuroblastoma and melanoma. The findings in this paper indicate that osteosarcomas express GD2 to a similar extent as neuroblastomas. These results would suggest clinical trials are worthwhile to further evaluate the benefit of anti-GD2 therapy in patients with osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5332. doi:10.1158/1538-7445.AM2011-5332

Lymphocyte Display: A Novel Antibody Selection Platform Based on T Cell Activation

Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69+ T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 103-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

Potent Anticarcinoma Activity of the Humanized Anti-CD70 Antibody h1F6 Conjugated to the Tubulin Inhibitor Auristatin via an Uncleavable Linker

The antitubulin agent monomethyl auristatin F (MMAF) induces potent antitumor effects when conjugated via protease cleavable linkers to antibodies targeting internalizing, tumor-specific cell surface antigens. Humanized 1F6 (h1F6) is a humanized monoclonal antibody targeting CD70, a member of the tumor necrosis factor family that is expressed on hematologic malignancies and carcinomas. Here, we tested h1F6-maleimidocaproyl (mc) MMAF conjugates, consisting of an uncleavable mc linker, for their ability to interfere with the growth of CD70-positive carcinomas. To evaluate the optimal drug per antibody ratio, we conjugated either four or eight MMAF molecules to the cysteines that comprise the interchain disulfides of h1F6 and determined antitumor activities in vitro and in xenografted mice. The tumor types tested included glioblastoma, patient-derived renal cell carcinoma (RCC) cell isolates, and standard RCC tumor cell lines. All h1F6-mcMMAF conjugates potently interfered with the growth of all carcinomas in vitro and resulted in complete responses of RCC tumors implanted orthotopically or s.c. in mice. In vitro, h1F6-mcMMAF(8) was generally more potent than h1F6-mcMMAF(4). However, h1F6-mcMMAF(4) displayed equal or better efficacy than h1F6-mcMMAF(8) when administered to tumor-bearing mice. We showed that h1F6-mcMMAF conjugates inhibited the growth of human carcinomas and that increased drug loading, while improving potency in vitro, did not substantially affect the pharmacodynamic and pharmacokinetic properties in vivo. Based on these findings, h1F6-mcMMAF(4), designated SGN-75, has been identified as a potential antibody-drug conjugate for clinical development.

Mapping tumor epitope space by direct selection of single-chain Fv antibody libraries on prostate cancer cells.

The identification of tumor-specific cell surface antigens is a critical step toward the development of targeted therapeutics for cancer. The epitope space at the tumor cell surface is highly complex, composed of proteins, carbohydrates, and other membrane-associated determinants including post-translational modification products, which are difficult to probe by approaches based on gene expression. This epitope space can be efficiently mapped by complementary monoclonal antibodies. By selecting human antibody gene diversity libraries directly on the surface of prostate cancer cells, we have taken a functional approach to identifying fully human, tumor-specific monoclonal antibodies without prior knowledge of their target antigens. Selection conditions have been optimized to favor tumor-specific antibody binding and internalization. To date, we have discovered >90 monoclonal antibodies that specifically bind and enter prostate cancer cells, with little or no binding to control cells. These antibodies are able to efficiently deliver intracellular payloads when attached to nanoparticles such as liposomes. In addition, a subset of the antibodies displayed intrinsic antiproliferative activity. These tumor-specific internalizing antibodies are likely to be useful for targeted therapeutics either alone or in combination with effector molecules. The antigens they bind constitute a tumor-specific internalizing epitope space that is likely to play a significant role in cancer cell homeostasis. Targeting components of this epitope space may facilitate development of immunotherapeutic and small molecule-based strategies as well as the use of other therapeutic agents that rely upon delivery to the interior of the tumor cell.

Diminution of antibodies directed against tumor cell surface epitopes: A single chain Fv fusion molecule specifically recognizes the extracellular domain of the c- erbB-2 receptor

We are evaluating strategies for the inhibition of growth or the selective killing of tumor cells. Cell surface antigens which are exclusively expressed or which are enhanced in their expression in tumor cells might provide the means to target cytotoxic or cytostatic agents to these cells. Few tumor specific cell surface antigens have been found, but the enhanced expression of growth factor receptors has been described for several types of tumors. A prominent example is the overexpression of the c- erbB-2 receptor in a high percentage of primary breast and ovarian carcinomas. We have derived monoclonal antibodies against the extracellular domain of the c- erbB-2 receptor. The antibody molecules were genetically engineered to minimize their size and to allow for their functional modification. For this purpose the cDNA sequences corresponding to the variable domains of one monoclonal antibody molecule (scFv) was expressed in bacteria and purified. We show in an immunoprecipitation experiment that this molecule retains its ability to recognize the c- erbB-2 extracellular domain. This molecule could become a valuable vehicle to specifically transport anti-tumor agents to breast cancer cells.

Recognition of rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): Studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: (i) Two clones showed H-2 restriction in tumor cell lysis, (ii) Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. (iii) One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells, (iv) One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells, (v) One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. (vi) Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen.

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.

No Expression of a Rous Sarcoma Virus-Induced Tumor Antigen in Mammalian Cells Infected With Retroviruses Transducing Other Oncogenes of the <italic>src</italic> Gene Family<xref ref-type="fn" rid="FN1">2</xref>

Immunization with mouse and rat cells transformed by Rous sarcoma virus (RSV) or by B77 avian sarcoma virus (ASV) induced complete transplantation resistance against an RSV-induced mouse tumor (CSA1M) in syngeneic hosts. In contrast, most of the mice immunized with a Fujinami sarcoma virus-transformed rat fibroblast line (FSV-3Y1), a feline sarcoma virus-transformed cat fibroblast line (FeSV-FEF), an Abelson leukemia virus-infected Balb/3T3 cell line (AbLV-3T3), or an uninfected 3Y1 cell line could not reject the CSA1M. Serologic analysis with the use of a complement-dependent cytotoxicity assay supported the results of transplantation studies. The mouse and rat cells transformed by RSV or B77 ASV expressed a common tumor-specific cell surface antigen (TSSA) detected by syngeneic antiserum against the CSA1M, whereas none of the FSV-3Y1, FeSV-FEF, and AbLV-3T3 cells expressed the TSSA. These results suggest that the common TSSA in the mouse and rat cells transformed by RSV or B77 ASV containing src gene is not shared with mammalian cells infected with retroviruses transducing other oncogenes of the src gene family (i.e., fps, fes, and abl).

Independent Expression of Common and Private Tumor-Specific Cell Surface Antigens in Somatic Cell Hybrids Between Rous Sarcoma Virus-Transformed and Normal Cells<xref ref-type="fn" rid="FN2">2</xref>

Somatic cell hybrids were prepared by fusion of a Rous sarcoma virus (RSV)-induced mouse tumor (CSA1M cl-11) with a Chinese hamster embryo lung fibroblast line (Wg3h X AGr X Ouar). Some hybrid clones showed rapid anchorage-independent growth and intracellular production of RSV-pp60src. Other hybrid clones showed slow anchorage-dependent growth and no production of such a protein. The former expressed both a common tumor-specific cell surface antigen(s) (TSSA) shared with other RSV-induced tumors and a private TSSA not shared with any other tumors; however, the latter lost the common TSSA and expressed only the private TSSA. These results suggest that the common and private TSSA on CSA1M cl-11 cells are independent genetic traits and only the common TSSA is coordinated with other expressions of the RSV src gene phenotype.

Correlation between tumor-specific surface antigens and src gene expression in Rous sarcoma virus-induced rat tumors

Immunization with Rous sarcoma virus (RSV)-induced mouse tumors or with SR-3Y1 or NY8-3Y1 rat fibroblasts transformed by a wild type RSV or an env gene deletion mutant of RSV induced complete transplantation resistance against an RSV-induced mouse tumor (CSA1M) in syngeneic hosts. On the other hand, 10 of 19 mice immunized with ts68-3Y1 rat fibroblasts transformed by an src gene temperature-sensitive mutant of RSV (permissive temperature: 33–35° C) could not reject the CSA1M. SR-3Y1, NY8-3Y1 and RSV-induced mouse tumors expressed a common tumor-specific cell-surface antigen (TSSA) detected by a syngeneic rat antiserum against NY8-3Y1. In ts68-3Y1, expression of the TSSA was temperature-sensitive, TSSA being detected only when ts68-3Y1 was cultivated at the permissive temperature. Immunoprecipitation showed that serum from a rabbit bearing an RSV-induced tumor detected a 60- kdalton protein in cell extracts from SR-3Y1 and NY8-3Y1. Antiserum to NY8-3Y1 failed to detect this protein. These results suggest that the tumor-specific surface antigen(s) on RSV-induced mammalian tumors was coded for by an RSV src gene that was not identical with the simple form of the 60- kdalton protein identified.

Individually Distinct Tumor-Specific Cell Surface Antigen Identified by Monoclonal Antibody on a Rous Sarcoma Virus-Induced Mouse Tumor<xref ref-type="fn" rid="fn2">2</xref>

Hybrid cell lines were prepared by the fusion of BALB/c myeloma P3U-1 cells with the lymphocytes of BALB/c mice that were immunized with syngeneic Rous sarcoma virus (RSV)-induced tumor CSA1M cells. Three clones of the hybrid progeny (3.4B2, 3.4C6, and 3.5C11) produced cytotoxic IgM antibodies against CSA1M cells. One of the clones, 3.5C11, was chosen for analysis of the detailed specificity. Both direct cytotoxicity assays and absorption tests revealed that monoclonal antibody from 3.5C11 was positive only with CSA1M cells and that it failed to react with other tumors, including 20 RSV-induced mouse tumors, and normal cells. The 3.5C11 monoclonal antibody alone, with or without exogenous complement, was suppressive in the therapy of ip injected CSA1M tumor in syngeneic hosts, and significant prolongation in survival was seen in the treated mice. These results clearly showed presence of an individually distinct tumor-specific cell surface antigen on an RSV-induced mouse tumor.