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  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
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Definitions

• This invention relates to fusion receptors for prostate-specific membrane antigen (PSMA), and to uses thereof in the treatment of prostate cancer, other cancers expressing PSMA and tumor neovasculature.
• PSMA prostate-specific membrane antigen
• the inventions provides fusion receptors, nucleic acids encoding these fusion receptors, and transduced cells expressing the fusion receptors, as well as methods of using the transduced cells.
• T cell immunity Destruction of immunological targets requires T lymphocyte recognition via the T cell receptor (TCR) of antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules on antigen- presenting cells (APC).
• TCR T cell receptor
• MHC major histocompatibility complex
• APC antigen- presenting cells
• T cell costimulation results from an interaction of the T cell surface receptor CD28 with the costimulatory hgand B7, which is p ⁇ maiily expressed on the surface of professional APCs and activated B cells leading to IL-2 secretion and clonal expansion of the activated T cells
• CD28 costimulatory hgand B7
• signals transduced by the CD28 receptor determine whether TCR occupancy results in a pioductive immune response or clonal anergy Therefore, one factor accounting for the pooi immunogenicity of MHC-expressmg tumors is that, despite presentation of potentially immunogemc peptides lithe context of MHC molecules, tumors lack the costimulatory molecule B7, and thus fail to elicit a full activation of T cells and therefore an effective anti-tumoi T cell response
• the introduction of the B7 molecule (CD28 hgand) in tumor cells is one discussed therapy today (melanoma Townsend et al, 1993, Chen et al, 1992
• T cells can lecogmze and lyse tumor cells provided that they bind to the tumor cells and are appiop ⁇ ately activated T cell activation operates according to the two signal model, which states that lymphocytes require for optimal activation both an antigen-specific signal delivered through the antigen receptor and a second antigen nonspecific or costimulatory signal T cell costimulatory pathways determine whether TCR complex engagement results in functional activation or clonal anergy of CD4 T cells
• T lymphocytes One means of generating tumor-specific T lymphocytes is then modification by gene transfer of tumor-specific fusion molecules
• the mtioduction of chime ⁇ c molecules m T cells combining tumor specific single chain variable fragment (scFv) with signal transduction domains of TCR related activation molecules is reported by a number of groups (Eshar et al, Springer Semin. Immunopathol. 18: 199-209 (1993)).
• scFv tumor specific single chain variable fragment
• These genetically modified T cells are able to target tumor cells and to destroy them in vitro, but based on the two signal model for T cell activation, the reinfusion of these transduced T lymphocytes is limited by the incomplete activation signal after antigen recognition and clonal expansion in vivo is not successful.
• antigen dependent IL-2 secretion can be stimulated in vitro in Jurkat cells expressing a chimeric molecule formed from an antigen-specific scFv and the ⁇ -chain of CD3, when the cells are exposed to the antigen in the presence of anti-CD28 or ionomycin as a costimulatory signal.
• Cells expressing both chimeric molecules displayed responses to either antigen in the presence of appropriate costimulatory molecules. Alvarez- Vallina et al. suggests that these results offer the possibility that addition of antigen-specific CD28 mediated signaling could improve adoptive immunotherapies.
• T-cell receptors have not been shown to function in human peripheral blood lymphocytes (PBL) and in particular in the T cells of actual cancer patients.
• PBL peripheral blood lymphocytes
• the present invention provides a fusion receptor composition which is effective to promote a cellular immune response to a target antigen in vivo when the fusion receptor is expressed by T lymphocytes.
• the target antigen is prostate-specific membrane antigen (PSMA)
• PSMA prostate-specific membrane antigen
• PSMA-scFv optional connector : cytoplasmic domain.
• the PSMA-scFv in this structure is a single chain antibody cloned from the V region genes of a hybridoma specific for PSMA.
• the optional connector region is provided to give a spacing between the PSMA-scFv and the cytoplasmic domain, such that both can retain substantial function.
• a suitable connector is the CD8 hinge, although other connectors of greater or lesser length might be used.
• the cytoplasmic domain is included to direct the function of the fusion receptor.
• One exemplary cytoplasmic domain which can be used in the fusion receptor of the invention is a T cell receptor ⁇ -chain cytoplasmic domain.
• an expression vector encoding the fusion receptor is transduced into primary T lymphocytes obtained from an individual to be treated, for example an expression vector encoding the PSMA-scFv containing fusion receptor is suitably transduced into cells from a human patient who has been diagnosed with prostate cancer.
• the transduced lymphocytes are returned to the patient where cells expressing the fusion receptor secrete interleukin 2 and proliferate in response to PSMA- positive cells.
• the resulting cytotoxic lymphocytes specifically lyse cells expressing PSMA and thus can be used to target PSMA-positive tumor cells.
• NK cells natural killer cells or other immune effector cells allows these cells to target any tissue (including tumor tissue) expressing PSMA.
• tissue including tumor tissue
• NK cells can be used to treat prostate cancer, other cancers expressing PSMA and tumor-associated neovasculature.
• Fig. 1 shows the structure of a retroviral vector Pz-1 including a gene for a PSMA-specific fusion receptor in accordance with the invention
• Figs. 2 A-E show cytotoxicity of Pz-1 transduced PBL with respect to various target cells
• Fig. 3 shows the time course for cocultivation of transduced T cells with fibroblasts
• Fig. 4A shows T cells proliferation in coculture with various types of fibroblast cells
• Fig. 4B shows cell lysis by T cells after prior exposure to coculture conditions
• Fig. 5 shows IL-2 production by transduced T cells in coculture with various types of fibroblast cells.
• the present invention provides fusion receptors which are useful in the generation of a cellular immune response to cells which express PSMA.
• fusion receptors have the general structure:
• PSMA-scFv optional connector : cytoplasmic domain. This structure is produced by expression in transduced cells of a DNA sequence encoding the ammo acid sequence of the fusion receptor
• PSMA-scFv is a single chain antibody cloned from the V region genes of a hybridoma specific for PSMA
• a suitable hybridoma for this purpose is J591, which is described m Liu et al , Cancer Res 57 3629-3635 (1997), although other hyb ⁇ domas which produce monoclonal antibodies specific to PSMA could also be employed The production of such hyb ⁇ domas has become routine, and the procedure will not be repeated here
• V H variable region heavy chain
• V L variable region light chain
• the cytoplasmic domain portion of the general formula set forth above is selected to enhance the characteristics of the fusion leceptoi for purposes of promoting a cellular immune response to the antigen recognized by the scFv portion of the fusion receptor
• the cytoplasmic domain is the cytoplasmic domain of a molecule which functions as a transducei of a mammalian immune response in the presence of an MHC- peptide complex or costimulatory factoi
• Representative, non-limitmg examples of cytoplasmic domains which may be employed m the present invention include the ⁇ -cham cytoplasmic domain, the CD28 cytoplasmic domain (particularly a fragment spanning ammo acids 336 to 663 of CD28 cDNA), 41BB, CD40, ICOS and trance
• the cytoplasmic domain is the ⁇ -chain derived the TCR complex
• the fusion receptor of the invention closely mimics a native TCR In this case, it might be expected that binding of an antigen to the scFv
• the fusion receptors of the invention may include othei cytoplasmic domains
• CD28 can be used as the cytoplasmic domain to enhance T-cell activation, survival and prohfeiation
• a preferred CD28 moiety is one which spans ammo acids 336 to 663 of CD28 cDNA, in which case no connector is needed to retain functon PSMA-fusion receptors incorporating 41BB as the cytoplasmic domain ha ⁇ e also been prepared
• Both the PSMA-CD28 and the PSMA-41 BB fusion receptors have been made and tested m the same experimental model used with the PSMA- ⁇ chain fusion receptor In both cases, sustained proliferation was obseived in both human CD4 and CD8 primary T cells (PBL) m the presence of PSMA ⁇ cells, with moie sustained proliferation being provided by the PSMA-41BB fusion receptor High production of IFN- ⁇ and IL-2 was observed, for PSMA-41BB and PSMA-CD28 transduced, respectively In each of the experiments performed
• the function of the connector is to act as a spacer so that both the scFv and the cytoplasmic domain can be functionally oriented within the membrane of the transduced cell.
• One exemplary connector is the CD8 hinge, although other connectors of greater or lesser length could be used. In some cases, such as using the CD28 fragment described herein, no connector is required to permit the molecules to assume the desired orientation.
• the chimeric fusion receptors are introduced into the individual to be treated (preferably a human) in one of two ways. Gene transfer can be carried out into bone marrow cells, either in vivo or ex vivo, or into immune effector/inflammatory cells such as T- lymphocytes or NK cells. Gene transfer may also be carried out into antigen presenting cells, particularly dendritic cells. In the case of dendritic cells, CD40 and trance are the preferred cytoplasmic domain.
• a preferred approach to this gene transfer is using retroviral vectors encoding the fusion receptor.
• a particularly preferred approach utilizes an SFG retroviral vector (Riviere et al., Proc. NatlAcad Sci. (USA) 92: 6733-6737 (1995)) transduced into patient PBL using gibbon ape leukemia virus (GALV) envelope-pseudotyped virions. (Gallardo et al., Blood 90: 952-957 (1997).
• the PSMA-specific fusion receptor of the present invention is useful in the treatment of prostate cancer.
• PSMA is also found in the neovasculature of renal cell, urothelial, colon, breast and lung carcinomas, melanomas and some sarcomas
• the PSMA-specific fusion receptor of the invention has broader applicability.
• the present application describes a method for treatment of cancers in which the cancer cells or neovasculature are characterized by expression of PSMA, comprising administering to a patient suffering from such a cancer patient-derived lymphocytes which express with a PSMA fusion receptor having the structure
• PSMA-scFv optional connector : cytoplasmic domain.
• administration is intended to encompass both in vivo methods, in which the fusion receptor is introduced into the lymphocytes without first removing them from the patient, and ex vivo methods where the patient-derived lymphocytes are obtained from the patient, transduced with the PSMA-specific fusion receptor and then reintroduced to the patient.
• the transduced lymphocytes are introduced in an amount to provide therapeutic benefit. Where sufficient clonal expansion of the transduced lymphocytes occurs in vivo, a long-term immunity to the tumor cells may be induced after a single administration. If the transduced lymphocytes are less stable, multiple infusions may be required to obtain remission of a particular cancer, and long-term protection may not be achieved. In either case, the determination of the appropriate therapeutic regimen is a matter of routine developed in the course of clinical trials.
• Example 1 PSMA-scFv was created by cloning the immunoglobulin genes from the J591 hybridoma encoding the variable region of the heavy chain (V H and the variable region of the light chain (V L ).
• the V H and V L genes were cloned using the technique previously described by Orlandi et al., supra. Briefly, mRNA was isolated from the J591 hybridoma cell line and reverse transcribed into cDNA using a reverse transcriptase polymerase chain reaction (RT- PCR) kit obtained from Pharmacia, Pisacatway, NJ.
• RT- PCR reverse transcriptase polymerase chain reaction
• V H gene Sequence analysis of the V H gene confirmed an appropriate open reading frame. However, the V L gene sequence analysis revealed a stop codon in the anticipated reading frame. The sequences of the genes encoding the J591 monoclonal antibody heavy and light chain were compared to the sequences of the cloned products, and several discrepancies were noted between the V L sequences. The major difference was that the primer pair used deleted a nucleotide from the actual sequence, resulting in an open reading frame shift that produced a stop codon. Nucleotide sequence corrections in the V L product were made using corrective primers based on the actual sequences and using these primers in a second PCR amplification utilizing the obtained V, sequence as a template. The corrective primers were: V L backward: GAAGAAGATCJG CATTGTGATGACCAGTCTC CAAATTCATG Seq. ID. No. 5
• an oligonucleotide encoding the human CD8 leader sequence was cloned to the 5'-end of the V H gene, and the 3'-end of the V,, gene was cloned to an oligonucleotide encoding a (gly-ser 2 ) 5 linker followed by the V L gene, creating the PSMA- specific scFv.
• the scFv was then cloned to the CD8 hinge and transmembrane domains, followed by the T cell receptor ⁇ -chain cytoplasmic domain to create Pz-1, a PSMA-specific scFv/ ⁇ -chain chimeric T-cell receptor.
• the Pz-1 fusion gene was then cloned into the SFG retroviral vector (Riviere et al, supra) as illustrated in Fig. 1.
• Example 2 The SFG retroviral vector containing Pz-1 was transduced into PBL's harvested from five human patients suffering from prostate cancer with a variety of clinical stages using GALV envelope pseudo typed virions as previously described. Gallardo et al., supra. The clinical status of three representative patients is summarized in Table 1.
• the age is the current age of the patient, the time since dx is the time elapsed between date of diagnosis of prostate cancer and PBL harvest; GG is the Gleason grade of the patient's most recent prostate cancer pathology; stage at dx is the clinical stage at the time of original diagnosis; RRP in the treatment column stands for radical retropubic prostatectomy; current stage is the current clinical or pathological state and current PSA is the most recent serum prostate specific antigen (PSA) level.
• PSA serum prostate specific antigen
• the PBL were expanded 4 to 14 days in the presence of interleukin-2 (IL-2).
• IL-2 interleukin-2
• Gene transfer efficiency was monitored by FACS analysis using a FITC-conjugated Pz-1 idiotype-specific antiserum. After incubation with the FITC labeled antiserum, the cells were washed incubated with 10% normal mouse serum, and stained with a PE-conjugated anti-CD8 mAb. The gene transfer efficiency observed varied between 20% and 50% in both CD8" and CD4 * cells for Pz-1 and controls.
• Example 3 Cytotoxic T lymphocyte (CTL) assays were performed on the human prostate cancer cell line LNCaP which abundantly expresses PSMA. In order to confirm that the cytotoxicity was PSMA-specific, PSMA was expressed in PC-3, a PSMA-negative human prostate cancer cell line and EL-4, a murine thymoma cell line. PBL from the three patients of Table 1 were transduced with either Pz-1 or NTP, a mutated human low-affinity nerve growth factor receptor used as a control cell surface marker. (Gallardo et al., supra). Transduction efficiency (%TR) measured as described above, and the fraction of CD8 + and CD56 + cells on the day of the CTL assay are reported in the common legend of Fig. 2.
• CTL Cytotoxic T lymphocyte
• E:T effector to target
• PBL transduced with Pz-1 but not NTP effectively lysed PSMA + targets.
• Example 4 To further assess the response of Pz-1 transduced primary T cells to PSMA, we investigated whether Pz-1 + PBL could undergo proliferation upon engagement with cell- bound PSMA and sustain thereafter their cytolytic potential.
• a cocultivation system was established in which transduced T cells were cultured with a layer of irradiated NIH3T3 fibroblasts expressing various combinations of PSMA and B7.1 for four days with periodic sampling to measure levels of IL-2 as illustrated in Fig. 3.
• FACS cell counts using FITC- conjugated Pz-1 idiotype specific antibody and with either anti-CD4 or anti-CD8 were performed after four days of co-cultivation, and again 4 days later.
• the transduced T cell count was derived by multiplying the percentage of CD4 + Pz-T or CD8 ⁇ Pz-l ⁇ double positive cells by the number of viable cells.
• the results are shown in Fig. 4A, where /B7+PSMA, /PSMA and /B7 refer to the molecules expressed by the fibroblast layer.
• PSMA induced proliferation of Pz-1 transduced T-cells increasing the number of cells 6-8 fold after 4 days, and these transduced cells destroyed PSMA + fibroblast layers within 48 hours, while the PSMA " layers remained intact during the entire 4-day cocultivation. By day 8, however, the absolute number of Pz-1 + cells dropped to 2-3 fold above initial levels.
• B7.1 (CD80) was transduced into PSMA + and PSMA " fibroblasts.
• Example 5 To test whether cytotoxic T cells retain their cytotoxic potential after restimulation with antigen, T cells were harvested 12-17 days after the start of coculture with fibroblast monolayers expressing PSMA and B7.1 and retested in the CTL assay. As shown in Fig. 5, expanded Pz-1 transduced T cells remained fully capable of lysing PSMA " target cells. Furthermore, the expanded Pz-1 + cells were capable of a second round of proliferation and IL-2 and IFN- ⁇ secretion when reexposed to PSMA + B7.1 + fibroblasts.
• T cells bearing artificial TCRs the prospect of apoptotic cell death or anergy upon restimulation could be compounded by partial T cell activation if chimeric receptors fail to adequately recruit downstream signaling molecules. Faulty T cell activation could result in the induction of immune tolerance and the neutralization of the infused effector T cells. Such phenomenon could in part explain in vivo findings obtained with T cells expressing an ErbB- 2 specific- ⁇ chain fusion receptor, required repeated high dose intra-tumoral administration to effectively eliminate established tumors. Alternschmidt et al, J. Immunol. 159: 5509-5515 (1997).
• Example 2 In order to prepare a fusion receptor in which the cytoplasmic domain is derived from CD-28, the procedure of Example 1 was repeated only using a CD28 fragment in place of the ⁇ -chain segment. The CD28 cDNA fragment was obtained as follow.
• a segment of the human CD28 cDNA that encodes part of the extracellular, the transmembrane, and the cytoplasmic domains was amplified by PCR from the plasmid pbsCD28, using the upstream primer
• primers contain Notl and BamHI sites respectively for the insertion of the PCR product in the retroviral Vector SFG.
• the CD28 fragment was ligated into the Notl an BamHI sites of the retroviral vector SFG, containing the CD8 ⁇ leader sequence, followed by the single chain gene, encoding the V H and V L domains of the PSMA-specific antibody

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