Aya Jakobovits

Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (κ) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 Vκ genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

Antigen–specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and κ light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen–specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.

The human immunoglobulin loci introduced into mice: V (D) and J gene segment usage similar to that of adult humans

Variable gene segments of the human immunoglobulin loci are represented in the human peripheral repertoire at different frequencies. XenoMouseTM strains contain approximately 2 megabases of the human immunoglobulin heavy and kappa light chain loci that functionally recapitulate the human humoral immune system. Analysis of human antibody transcripts from XenoMouse spleens and lymph nodes revealed that V, D and J gene segment utilization from these unimmunized animals were nearly identical to the gene segment utilization reported for humans with extensive antigenic histories.

Production of Antigen-Specific Human Antibodies from Mice Engineered with Human Heavy and Light Chain YACsa

Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies. The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes. This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration. The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation. Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies. Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum. These results suggest the potential value of these mice as a source of fully human antibodies for human therapy. Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy. Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization. The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders. The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

Monoclonal Antibodies to Six-Transmembrane Epithelial Antigen of the Prostate-1 Inhibit Intercellular Communication In vitro and Growth of Human Tumor Xenografts In vivo

Six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) is a novel cell surface protein highly expressed in primary prostate cancer, with restricted expression in normal tissues. In this report, we show STEAP-1 expression in prostate metastases to lymph node and bone and in the majority of human lung and bladder carcinomas. We identify STEAP-1 function in mediating the transfer of small molecules between adjacent cells in culture, indicating its potential role in tumor cell intercellular communication. The successful generation of two monoclonal antibodies (mAb) that bind to cell surface STEAP-1 epitopes provided the tools to study STEAP-1 susceptibility to naked antibody therapy. Both mAbs inhibited STEAP-1-induced intercellular communication in a dose-dependent manner. Furthermore, both mAbs significantly inhibited tumor growth in mouse models using patient-derived LAPC-9 prostate cancer xenografts and established UM-UC-3 bladder tumors. These studies validate STEAP-1 as an attractive target for antibody therapy in multiple solid tumors and provide a putative mechanism for mAb-induced tumor growth inhibition.

Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells

With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (VH) genes, the major diversity (D) gene cluster, the joining (JH) genes, the intronic enhancer (EH), and the constant region genes, mu (C mu) and delta (C delta). Two IgK locus-derived YACs each contain three variable (V kappa) genes, the joining (J kappa) region, the intronic enhancer (E kappa), the constant gene (C kappa), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form.

Plucking of lectin receptors from erythrocytes: Isolation of cell surface components without the use of detergents

Abstract A new approach is described for the isolation of lectin receptors without the use of detergents, by plucking them from the cell surface. Cells bound to lectin-coated Sepharose beads are sheared off the beads by mechanical disruption, whereupon the receptors remain attached to the beads and are released specifically by inhibitory sugars. Material plucked from neuraminidase-treated human erythrocytes by beads coated with peanut agglutinin and released by D-galactose was identified as asialoglycophorin. The same membrane glycoprotein was plucked from neuraminidase-treated erythrocytes by beads coated with soybean agglutinin, but at considerably lower yield.

SPECIFIC LYSIS OF ANTIGENICALLY IRRELEVANT CELLS BY CYTOTOXIC T LYMPHOCYTES UPON INSERTION OF APPROPRIATE ANTIGENS INTO THE TARGET CELL PLASMA MEMBRANES

The importance of the membrane milieu to functional presentation of target cell (TC) antigens to cytotoxic T lymphocytes (CTL) was investigated by examining the interaction of CTL with TC plasma membrane (PM) fractions, in isolated form or integrated into antigenically irrelevant TC. Isolated ascitic vesicles, microsomes, and purified PM, containing serologically defined alloantigens that have been implicated as the relevant TC antigens, effectively, yet nonspecifically, inhibited the binding and lysis of TC by CTL. The same PM fractions, when inserted into antigenically irrelevant TC via vesicles containing Sendai virus components, rendered the TC susceptible to CTL-mediated lysis directed against the inserted antigens. These findings suggest that CTL interact specifically with TC determinants only when they are embedded in the proper membrane environment.

Abstract 4393: ASG-5ME is a novel antibody drug conjugate (ADC) for treating prostate cancers

AGS-5 is a novel transmembrane antigen discovered by Agensys using transcriptional profiling that is highly expressed in a variety of epithelial tumors. A fully human IgG2k monoclonal antibody that binds to AGS-5 with high affinity was conjugated with the anti-microtubulin drug, monomethyl auristatin E (MMAE), via a conditionally labile valine-citrulline (vc) maleimidocaproyl linker to yield an average drug: antibody ratio of 3.7. Following cell surface binding, ASG-5ME is internalized and trafficked through the endocytic pathway, where proteolytic cleavage releases free active drug that subsequently binds and disrupts microtubules. We evaluated the expression of AGS-5 protein in a large number of prostate patient tumors using IHC. ASG-5ME was evaluated for its cell cytoxicity in vitro, and its anti-tumor activity was investigated on different established xenograft tumors, using both patient-derived and cell line models of prostate cancer. The pharmacokinetics of ASG-5ME in mice was also evaluated. Our studies indicated AGS-5 protein was expressed in more than 95% of primary prostate cancer specimens and distant metastases, and on more than 90% and 80% of pancreatic and gastric cancer specimens, respectively. ASG-5ME, bound with high affinity (Kd = 0.5 nM) to both human and cynomolgus monkey AGS-5 and mediated potent dose dependent cell cytotoxicity in vitro. ASG-5ME showed significant long term tumor regression and eradication of multiple established prostate cancer xenografts, including those grown orthotopically. Phamacokinetic (PK) analysis of ASG-5ME in mice indicated that the ADC was stabile with long T1/2 of ∼12 days. When considered together these data indicate that ASG-5ME is a promising therapeutic ADC for the treatment of prostate and other AGS-5 expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4393.

Abstract 2590: Preclinical evaluation of ASG-5ME, a novel antibody-drug conjugate for pancreatic cancer

Antibody-drug conjugates (ADCs) have demonstrated promising activity and tolerability profiles in oncology clinical trials, including for Hodgkin lymphoma (brentuzumab vedotin) and breast cancer (trastuzumab-DM1), motivating the development of new ADCs targeting antigens in other prevalent cancers. An immunohistochemical survey of expression of the novel tumor antigen AGS-5 revealed strong staining in 90% of pancreatic cancer specimens tested. Importantly, this staining was largely uniform, with essentially all transformed cells demonstrating membranous staining. This observation along with the observation of equally abundant expression of AGS-5 in prostate and other cancers motivated the development of ASG-5ME, an ADC targeting AGS-5. The ADC is comprised of a fully human IgG2 monoclonal antibody conjugated to the potent tubulin-binding drug monomethylauristatin E, conjugated with an average of 3.7 drug molecules per antibody. Conjugation and analysis of this IgG2 ADC will be discussed. The resulting conjugate demonstrated antigen-dependent internalization and in vitro cytotoxicity at sub-saturating ADC concentrations, as well as potent in vivo antitumor activity in patient-derived xenograft models of pancreatic cancer. ASG-5ME has a long (12 day) T1/2 in mice. Taken together, these results support the clinical evaluation of ASG-5ME for treatment of pancreatic cancer Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2590.