Tumor Cell Surface Antigens Research Articles

Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs). First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells.

Targeted Nanomaterials for Radiotherapy

Nanomaterials have garnered increasing interest recently as potential therapeutic drug-delivery vehicles. Among the existing nanomaterials are the pure carbon-based particles, such as fullerenes and nanotubes, various organic dendrimers, liposomes and other polymeric compounds. These vehicles have been decorated with a wide spectrum of target-reactive ligands, such as antibodies and peptides, which interact with cell-surface tumor antigens or vascular epitopes. Once targeted, these new nanomaterials can then deliver radioisotopes or isotope generators to the cancer cells. Here, we will review some of the more common nanomaterials under investigation and their current and future applications as drug-delivery scaffolds with particular emphasis on targeted cancer radiotherapy.

Clinico-pathological significance of RCAS1 expression in gliomas: A potential mechanism of tumor immune escape

RCAS1, one of the tumor cell surface antigens, is strongly expressed in aggressive tumors. RCAS1 suppresses the in vitro growth of immune effector cells. We investigated the expression of RCAS1 in 57 gliomas using immunohistochemistry. Furthermore, we examined the association of the RCAS1 expression with the infiltration of tumor infiltrating lymphocyte (TIL). RCAS1 overexpression was significantly correlated with high histological grade and poor prognosis. Reduced infiltration and increased apoptosis of TILs was observed in RCAS1-positive regions. Apoptotsis of TILs appeared to be induced by RCAS1. RCAS1 expression in gliomas may play roles in tumor progression and tumor immune escape.

Development of a novel strategy for engineering high-affinity proteins by yeast display

Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.

Heteroconjugated antibodies enhance lymphocyte-mediated tumour cell lysis in vitro and in vivo.

Covalent linkage of an antitumour antibody specific for a tumour cell surface antigen to an antilymphocyte antibody specific for the T lymphocyte receptor complex produces a heteroconjugated antibody that can activate and redirect cytotoxic T lymphocytes to lyse tumour cells. The ability of an antilymphocyte-antitumour heteroconjugate (500A2 x 96.5) to direct the lysis of murine melanoma cells by cultured murine lymphocytes was tested in vitro using a 4-h chromium release assay and in vivo with a tumour neutralization assay. In vitro, the addition of heteroconjugated antibody significantly increased tumour lysis by murine C3H/HeN lymphocytes (median specific lysis 82.7 per cent with lymphocytes plus heteroconjugate versus 9.5 per cent for lymphocytes alone, P less than 0.001). In vivo, treatment with heteroconjugated antibody plus lymphocytes significantly reduced the development of pulmonary metastases after intravenous tumour administration (median number of pulmonary metastases 28.5 for combined treatment versus 250 for heteroconjugate or lymphocytes alone, P less than 0.001).

Radioimmunotargeting of Human Rhabdomyosarcoma Using Monoclonal Antibody 8H9

Although metastatic rhabdomyosarcoma (RMS) is chemotherapy and radiotherapy responsive, few patients are cured. 8H9, a murine IgG(1) monoclonal antibody, recognizes a unique cell surface tumor antigen broadly distributed on neuroectodermal, epithelial, and mesenchymal tumors, including RMS. We now report on the in vitro characterization of radiolabeled 8H9 and its in vivo immunotargeting potential in mice with subcutaneous human RMS. Saturation-binding studies carried out to determine (125)I-8H9 affinity to the RMS cell line HTB82 demonstrated that (125)I-8H9 had a K(d) of 10.3nM with an estimated 115,000 binding sites on every HTB82 cell. (125)I-8H9 was retained on the cell surface without significant internalization. Biodistribution of (125)I-8H9 was studied in athymic mice bearing HTB82 xenografts. Following intravenous injection of 4.44MBq of (125)I-8H9, selective tumor uptake was evident 4 to 172 hours after injection. Average tumor uptake was 7.0 +/- 1.8, 11.5 +/- 3.9, 15.1 +/- 3.7, and 5.4 +/- 1.2% injected dose per gram at 4, 24, 48, and 172 hours, respectively. Mean tumor: tissue ratios were maximal at 172 hours (for lung, 4, kidney, 6, liver, 7, spleen, 11, femur, 14, muscle, 18, and brain, 48). Established RMS xenografts treated with a single injection of 18.5 MBq (131)I-8H9 were significantly suppressed compared to controls. Radiolabeled 8H9 effectively targeted RMS xenografts and may have a potential clinical role in radioimmunotherapy.

797. Targeted Elimination of Prostate Cancer by Genetically Directed Human T Lymphocytes

The genetic engineering of T cells is a novel strategy designed to accelerate the generation of tumor-specific T cells and remedy the biological limitations that constrain the anti-tumoral functions of normal T cells. Chimeric antigen receptors (CARs) are essential constituents of this new armamentarium. Unlike the physiological TCR, CARs encompass immunoglobulin variable regions or receptor ligands as antigen-recognition elements, thus permitting T cells to recognize cell surface tumor antigens in the absence of HLA expression. T cell activation is mediated by the CARs cytoplasmic domain, typically derived from the CD3z chain or the FcRIgchain. The signaling function of CARs has been questioned based on findings in transgenic mice and transduced primary T cells indicating that antigen ligation was not sufficient to activate resting naive mouse T cell or elicit IL2 secretion in retrovirally transduced human peripheral blood T cells. We and others subsequently showed that z chain-based CARs could induce strong activation capable of sustaining T cell proliferation and permitting secondary antigenic restimulation in vitro, provided that antigen was presented in the context of CD28-mediated costimulation. It is not known, however, whether T cells expanded in this manner, particularly human T cells, could mediate tumor eradication in vivo, and whether further in vivo costimulation would be needed to sustain their function. Here we investigate the in vivo function of Pz1, a CAR targeting human PSMA. PSMA is a highly attractive target antigen that is found in most prostate cancer cells, including hormone refractory metastatic disease. The Pz1 receptor encompasses the z chain of the CD3 complex as its activation domain and specifically redirects in vitro cytolysis of retrovirally transduced peripheral blood T lymphocytes (PBLs) against PSMA-positive tumor cell lines. To investigate whether expanded Pz1+ T cells are active in vivo and require costimulation after adoptive transfer, we established three tumor models in SCID-bg/bg mice- orthotopic, subcutaneous and pulmonary. Direct administration of Pz1+ T cells in orthotopic and subcutaneous human prostate tumors ranging in size from 0.04 to 0.28 cm3 eliminated a majority of the tumors. T cells transduced with a control receptor had no anti-tumor effect. We next developed a systemic tumor model using a murine tumor expressing human PSMA in which mice succumbed to pulmonary tumors in 14 days. Intravenous administration of Pz1-transduced T induced objective responses in all mice and cured a substantial fraction of them. Interestingly, lung nodules continued to increase in size for several days after T cell infusion, before regressing completely. This finding demonstrates the critical importance of activating T cells so that they survive and sustain their function for at least 7-10 days after infusion. Altogether, our results strongly support the feasibility of targeting prostate cancer with autologous T lymphocytes directed against prostate-specific membrane antigen by a z chain-based receptor.

DNA vaccines to attack cancer.

Delivery of antigens by injection of the encoding DNA allows access to multiple antigen-presenting pathways. Knowledge of immunological processes can therefore be used to modify construct design to induce selected effector functions. Expression can be directed to specific intracellular sites, and additional genes can be fused or codelivered to amplify responses. Therapeutic vaccination against cancer adds a requirement to overcome tolerance and to activate a weakened immune repertoire. Induction of CD4(+) T helper cells is critical for both antibody and T cell effector responses. To activate immunity against tumor antigens, we fused the tumor-derived sequences to genes encoding microbial proteins. This strategy engages T helper cells from the large antimicrobial repertoire for linked help for inducing antibody against cell-surface tumor antigens. The principle of linked T cell help also holds for induction of epitope-specific antitumor CD8(+) T cells, but the microbial sequence has to be minimized to avoid competition with tumor antigens. Epitope-specific DNA vaccination leads to powerful antitumor attack and can activate immunity from a profoundly tolerized repertoire. Vaccine designs validated in preclinical models are now in clinical trial with immune responses detected against both tumor antigens and fused microbial antigens. DNA priming is highly efficient, but boosting may benefit from increased antigen expression. Physical methods including electroporation provide increased expression without introducing additional competing antigens. A wide range of cancers can be targeted, and objective assays of response will determine efficacy.

Tumor-specific Ab-mediated targeting of MHC-peptide complexes induces regression of human tumor xenografts in vivo.

A cancer immunotherapy strategy is described herein that combines the advantage of the well established tumor targeting capabilities of high-affinity recombinant fragments of Abs with the known efficient, specific, and potent killing ability of CD8 T lymphocytes directed against highly antigenic MHC-peptide complexes. Structurally, it consists of a previously uncharacterized class of recombinant chimerical molecules created by the genetic fusion of single-chain (sc) Fv Ab fragments, specific for tumor cell surface antigens, to monomeric scHLA-A2 complexes containing immunodominant tumor- or viral-specific peptides. The fusion protein can induce very efficiently tumor cell lysis, regardless of the expression of self peptide-MHC complexes. Moreover, these molecules exhibited very potent antitumor activity in vivo in nude mice bearing preestablished human tumor xenografts. These in vitro and in vivo results suggest that recombinant scFv-MHC-peptide fusion molecules could represent an approach to immunotherapy, bridging Ab and T lymphocyte attack on cancer cells.

HLA DR-directed bispecific single-chain Fv antibodies for lymphoma therapy

Fc receptors are important for the clinical efficacy of therapeutic antibodies. Bispecific antibodies (BsAb) are immunoglobulin-conjugates with two different binding specifities, targeting tumor antigens and effector cell trigger molecules. BsAb, produced by chemical coupling of one antibody against a tumor cell surface antigen with another against a Fc receptor, mediate effective interactions between effector and target cells.

Immunotoxins - a new class of anticancer drugs

Immunotoxins deliver a toxin specilically to tumor cells by targeting tumor cell-surface antigens, without harming the narmal cell. Once inside the cell, the toxin kills the turner cell by either inhibiting protein synthesis or altering signal transduction pathmays. Immunotoxins improve the therapeutic index, with the potential to enhance efficacy and decrease systemic toxicity. Several immunoloxins have shown impressive anti-cancer activity in various stages of clinical trials. We present a brief overview of recent progress in the clinical development of immunotoxins.

Chimeric TNT-3/human beta-glucuronidase fusion proteins for antibody-directed enzyme prodrug therapy (ADEPT).

ADEPT (antibody-directed enzyme prodrug therapy) is a novel therapeutic approach that targets an enzyme into tumors to convert a relatively nontoxic prodrug into an active cytotoxic agent. This method has a number of advantages, including the reduction of systemic toxicity, but to date it has not realized its full potential. A critical component of ADEPT is the choice of the monoclonal antibody (MAb) to target the enzyme into the tumor mass. Prior studies have utilized MAbs directed against tumor cell surface antigens which are oftentimes labile and heterogeneous in nature and do not provide an ideal site for the enzyme. As an alternative approach, we now describe the use of Tumor Necrosis Therapy (TNT) MAbs to deliver the enzyme to necrotic regions of tumors in order to enhance the effectiveness of ADEPT. Biodistribution and autoradiographic studies performed using TNT MAbs have shown that localization of these antibodies occurs in degenerating cells and necrotic regions of tumors and that binding is retained within the tumor mass for extended periods of time. Since necrotic regions are often located in the center of tumors, are universal in nature, and constitute between 30 and 80% of the tumor mass, TNT MAbs may be ideal targeting agents for ADEPT. To test this hypothesis, fusion proteins consisting of single chain Fv (scFv), Fab, or F(ab')2 fragments of chTNT-3 and the human beta-glucuronidase (betaG) enzyme were constructed for ADEPT. Each of these reagents was tested to assess specificity and avidity of antigen binding as compared to the parental antibody. In addition, studies were performed to demonstrate enzymatic function of the fusion proteins and retention of catalytic activity in circulating blood, specific tissues, and tumor after in vivo administration. Pharmacokinetic and biodistribution studies of radiolabeled fusion proteins were conducted over time to evaluate the characteristics of the fusion proteins. Finally, one of the constructs (chTNT-3 Fab/betaG) was used in a pilot treatment study with a glucuronide prodrug of doxorubicin to demonstrate the anti-tumor activity of ADEPT using the chemoresistant MAD109 murine lung carcinoma tumor model transplanted into BALB/c mice. The results of these experiments show that all three constructs retained their antigen binding capability and demonstrated active enzymatic function against substrate in vitro. Moreover, after in vivo administration, the betaG enzyme was shown to localize to tumor and remain active for up to 9 days demonstrating a key characteristic of TNT targeting. Pharmacokinetic and biodistribution studies confirmed specific localization of the fusion proteins and rapid clearance from blood and normal tissues over time. Finally, therapeutic studies using only two doses of fusion protein followed by prodrug administration demonstrated active cytotoxicity against established tumors without systemic toxicity. These preliminary studies show that the use of TNT MAbs to target the enzyme to the tumor may be a significant advance in ADEPT and that further studies are warranted to test this novel therapeutic approach in the treatment of solid tumors.

Strategies for vascular targeting in tumors.

Strategies for vascular targeting in tumors.

Cloning, isolation and characterization of human tumor in situ monoclonal antibodies.

A bacterially expressed human antibody (Ab) library (diversity approximately 10(5)) was generated from tumor-infiltrating B lymphocytes present in tissue isolated from a colon tumor. Immunoglobulin (IgG) heavy and light chain variable regions were amplified without isolating or enriching B cells, cloned into a phage-expression vector, and soluble antigen-binding fragment (Fabs) from >10(5) members of the library were screened rapidly by two distinct and complementary methodologies. In the first approach, soluble Fabs were screened by enzyme-linked immunosorbent assay (ELISA) on tumor cell monolayers. Alternatively, tumor cell surface antigens were selectively biotinylated with a plasma membrane-impermeable reagent, solubilized with non-ionic detergent, and were used to screen >10(5) members of the Ab library by capture lift. Reactive Fabs were partially characterized for tumor cell specificity and cross-reactivity, resulting in the identification of multiple Abs that bind cultured tumor cells but not normal human fibroblasts. The Fabs clustered into at least three distinct epitope specificity groups based on multiple criteria, including differential reactivity on two tumor cell lines and distinct antigen recognition patterns on western blot and immunoprecipitation. Moreover, DNA sequencing of the Ab variable regions demonstrated that the majority of the tumor-reactive Fabs were distinct and substantially different from the corresponding most homologous Ab germline gene. The relatively small size of the tumor-derived library allowed direct screening of soluble Fab of every member of the library, permitting the characterization of multiple human monoclonal antibodies (mAbs) that might not be discovered using alternative approaches, such as hybridoma technology or phage-display.

Characterization of a phage display-derived human monoclonal antibody (NHS76) counterpart to chimeric TNT-1 directed against necrotic regions of solid tumors.

To eliminate the human anti-mouse antibody (HAMA) response seen in patients treated with murine and chimeric antibodies, fully human monoclonal antibodies (MAbs) are now being developed. Tumor Necrosis Therapy (TNT) is an approach to tumor targeting that utilizes MAbs directed against common intracellular antigens such as nucleic acids, accessible only in necrotic areas of solid tumors. By binding to the necrotic core of tumors, these TNT MAbs can circumvent many of the limitations of MAbs directed against tumor cell surface antigens. Chimeric TNT-1 (chTNT-1) was first developed from the parent murine antibody by genetically engineering the murine variable regions to the human IgG(1) and kappa constant regions. Although the chimeric antibody's behavior was similar to that of the murine version, the 35% murine homology it shares allows for the potential of a HAMA response. A human antibody derived from a phage display library, designated NHS76, has been developed with similar binding characteristics to the TNT-1. To demonstrate that this genetically engineered human counterpart to chTNT-1 has similar pharmacokinetic characteristics, in vivo behavior, and targeting abilities, both antibodies were rigorously tested in parallel. For these studies, biodistribution analysis in LS174T human colon tumor-bearing nude mice was performed to compare the uptake levels in tumor and normal organs. In addition, mouse imaging and autoradiographic studies were conducted to demonstrate positive uptake in necrotic regions of tumor and negative uptake in viable tissues and organs. The results of these studies confirm the comparable nature of both antibodies and provide the necessary preclinical data to show the suitability of NHS76 as an improved product for the therapy of solid tumors in man.

Dissection and optimization of immune effector functions of humanized anti-ganglioside GM2 monoclonal antibody

A mouse/human chimeric monoclonal antibody (MAb) KM966, specific for the cell-surface tumor antigen ganglioside GM2, was humanized by the complementarity determining regions (CDRs) grafting method. Not only the amino acid residues in the CDRs but also several in the framework regions (FRs) were changed from the human to the murine residues. A humanized variant, huKM796H/Lm-28, containing eight and five amino acid alterations in variable light (VL) and variable heavy (VH) FRs, respectively, showed a 9-fold reduction in complement-dependent cytotoxicity (CDC) compared to the chimeric KM966, despite tight antigen binding and potent antibody-dependent cellular cytotoxicity (ADCC). Several additional variants were subsequently constructed to improve the CDC of the antibody. One of the variants, designated KM8969, which differs by three amino acids, exhibited a CDC within 3-fold of the chimeric KM966. In addition, humanized KM8969 bound GM2 antigen 1.25-fold more tightly than the chimeric KM966 and showed 5-fold higher ADCC than the chimeric KM966. These results clearly show that the humanized KM8969, having the optimized immune effector functions and theoretically minimal immunogenicity, is an ideal candidate to test the effectiveness of anti-GM2 MAb in human cancer therapy. Taken together, the results obtained here indicate that the ADCC and CDC of an antibody can be dissected independently via engineering of the antibody variable region.

STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors.

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell-cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.

Neural cell surface differentiation antigen gp130(RB13-6) induces fibroblasts and glioma cells to express astroglial proteins and invasive properties.

Transient expression of the differentiation and tumor cell surface antigen gp130(RB13-6) characterizes a subset of rat glial progenitor cells susceptible to ethylnitrosourea-induced neurooncogenesis. gp130(RB13-6) is as a member of an emerging protein family of ecto-phosphodiesterases/nucleotide pyrophosphatases that includes PC-1 and the tumor cell motility factor autotaxin. We have investigated the potential role of gp130(RB13-6) in glial differentiation by transfection of three cell lines of different origin that do not express endogenous gp130(RB13-6) (NIH-3T3 mouse fibroblasts; C6 and BT7Ca rat glioma cells) with the cDNA encoding gp130(RB13-6). The effect of gp130(RB13-6) expression was analyzed in terms of overall cell morphology, the expression of glial cell-specific marker proteins, and invasiveness. Transfectant sublines, consisting of 100% gp130(RB13-6)-positive cells, exhibited an altered, bipolar morphology. Fascicular aggregates of fibroblastoid cells subsequently developed into mesh-like patterns. Contrary to the parental NIH-3T3 and BT7Ca cells, the transfectant cells invaded into collagen type I. As shown by immunofluorescence staining of the transfectant sublines as well as of primary cultures composed of gp130(RB13-6)-positive and -negative cells, expression of gp130(RB13-6) induced coexpression of proteins typical for glial cells and their precursors, i.e., glial fibrillary acidic protein, the low affinity nerve growth factor receptor, and the neural proteins Thy-1, Ran-2, and S-100. In accordance with its expression in the immature rat nervous system, gp130(RB13-6) may thus have a significant role in the glial differentiation program and its subversion in neurooncogenesis.

The Role of Homophilic Binding in Anti-tumor Antibody R24 Recognition of Molecular Surfaces: DEMONSTRATION OF AN INTERMOLECULAR β-SHEET INTERACTION BETWEEN VH DOMAINS

The murine antibody R24 and mouse-human Fv-IgG1(kappa) chimeric antibody chR24 are specific for the cell-surface tumor antigen disialoganglioside GD3. X-ray diffraction and surface plasmon resonance experiments have been employed to study the mechanism of "homophilic binding," in which molecules of R24 recognize and bind to other molecules of R24 though their heavy chain variable domains. R24 exhibits strong binding to liposomes containing disialoganglioside GD3; however, the kinetics are unusual in that saturation of binding is not observed. The binding of chR24 to GD3-bearing liposomes is significantly weaker, suggesting that cooperative interactions involving antibody constant regions contribute to R24 binding of membrane-bound GD3. The crystal structures of the Fabs from R24 and chR24 reveal the mechanism for homophilic binding and confirm that the homophilic and antigen-binding idiotopes are distinct. The homophilic binding idiotope is formed largely by an anti-parallel beta-sheet dimerization between the H2 complementarity determining region (CDR) loops of two Fabs, while the antigen-binding idiotope is a pocket formed by the three CDR loops on the heavy chain. The formation of homophilic dimers requires the presence of a canonical conformation for the H2 CDR in conjunction with participation of side chains. The relative positions of the homophilic and antigen-binding sites allows for a lattice of GD3-specific antibodies to be constructed, which is stabilized by the presence of the cell membrane. This model provides for the selective recognition by R24 of cells that overexpress GD3 on the cell surface.

Discovery of Human Antibodies to Cell Surface Antigens by Capture Lift Screening of Phage-Expressed Antibody Libraries

An assay for the rapid identification and cloning of antibody fragments (Fabs) reactive with cell surface antigens was established and used to identify Fabs selectively reactive with tumor cell surface antigens. The Fabs were produced by a phage expression system and screened by a modified plaque lift approach in which nitrocellulose filters were coated with an anti-immunoglobulin reagent and blocked with bovine serum albumin prior to application to the phage-infected bacterial lawn. Subsequently, capture lifts were incubated with biotinylated antigen and reactive Fabs were identified with streptavidin conjugates. This screening method, termed capture lift, results in the immobilization of greater quantities of Fab and decreases the binding of unrelated host proteins, resulting in a more sensitive plaque lift assay. The capture lift permits the simulataneous analysis of thousands of antibody clones and, more importantly, can be used with crude detergent-solubilized cell extracts, permitting the discovery of Fabs which bind integral membrane proteins present in heterogeneous mixtures of antigens. Optimal conditions were identified utilizing phage-expressed BR96 Fab and a horseradish peroxidase conjugate of Lewis Y, a soluble cross-reactive antigen. Subsequently, it was demonstrated that the assay was functional with postnuclear detergent extracts isolated from surface-biotinylated tumor cells expressing the BR96 tumor antigen. Purification of the target antigen was not required. To demonstrate the application of the capture lift assay for the discovery of Fabs reactive with novel cell surface antigens a phage-expressed human antibody library constructed from tumor-infiltrating B lymphocytes was screened. Multiple antibody clones which reacted with detergent-solubilized biotinylated surface antigens were identified. Upon further characterization a portion of these displayed selectivity for tumor cells, as demonstrated by the binding of Fab to fixed and live tumor cells but not normal fibroblasts.