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  • Coley’s early studies led to the use of bacilleCalmette-Guérin (BCG) for cancer immunotherapy,Achieved rate of up to 10% for sarcoma patients.Streptococcus pyogenes and Serratiamarcescens , together with the still-active endotoxins directly into tumors
  • Peptides from three normal self proteins (yellow, blue, and green) are presented on the cell surface as normal self peptides (yellow, blue, and green) in major-histocompatibility-complex (MHC) molecules. In cases of mutation (Panel A), failure of the tumor cell to re- pair DNA damage can result in a mutation (red) in a normal protein and, consequently, presentation of mutated peptides (red) on the surface of tumor cells. Because of a mutation or factors that regulate its expression, a normal protein (green) can be overexpressed in a tumor cell and its peptides presented on the cell surface at highly abnormal levels (Panel B). In cases of post-translational modification (Panel C), a normal protein can be abnormally processed (spliced, glycosylated, phosphorylated, or lipidated) post-translationally (green stripes), resulting in an abnormal repertoire of peptides on the surface of the tumor cell.
  • Tumor-infiltrating lymphocytes (TILs) in melanoma are responsible for tumor cell killing and may induce spontaneous regression. Brisk TILs in the melanoma vertical growth phase is a strong, albeit not independent, prognostic factor associated with superior survival. In a study of 1,171 patients with cutaneous vertical growth phase melanoma with at least 3 years of follow-up, we investigated whether more detailed classes of TIL patterns, based on topography and intensity, are independent prognostic factors and identify patients with good prognosis (Spatz et al., 2007). TIL infiltrate was assessed for pattern (absent, non brisk, brisk), intensity (scanty, moderate, dense), and topography (peripheral, central, both). Pattern was assessed qualitatively: brisk TIL infiltrate was defined as a continuous band of lymphocytes at the base of the melanoma, or throughout the tumor. Other studied variables were: thickness, mitotic count, ulceration, gender, age, anatomic site, and melanoma-related death (MRD). A brisk infiltrate was observed in 21.2% of the cases. Adjusted hazard ratio for MRD as compared to the absent category was 0.82 (95% CI=0.64-1.06) in the non-brisk category, and 0.43 (95% CI=0.28-0.68) in the brisk category. Based on the Cox model, the presence of brisk TILs was an independent prognostic factor for MRD (p<0.001), controlling for thickness, mitotic count, ulceration, gender, age and site. Intensity of the TIL infiltrate was significantly associated with MRD for melanomas with peripheral and central brisk TILs (p=0.027) but not for other melanomas. Remarkably, no death was observed in the 5.5% of melanomas with dense, brisk TIL infiltrate at ten years. Therefore, it is likely that there is a small population of “super-responder” patients whose tumor is characterized by a continuous and dense TIL infiltrate and who do not display signs of tumor progression. In the context of the recent data showing a therapeutic effect of ipilimumab anti-CTLA4 therapy in patients with metastatic melanoma, it is of utmost importance to study whether the amount of regulatory T cell infiltrate in the primary tumor or sentinel node is a predictive factor for response. Although it has been reported that ex vivo expansion of TIL infiltrate does not expand the regulatory T cell compartment (Knol et al., 2008), the interaction between TILs and regulatory T cells needs to be better understood.
  • Following activation with antigen, B cells begin to proliferate rapidly. In these rapidly dividing cells, the genes encoding the variable domains of the heavy and light chains undergo a high rate of point mutation, by a process called somatic hypermutation (SHM). SHM results in approximately one nucleotide change per variable gene, per cell division.[8] As a consequence, any daughter B cells will acquire slight amino acid differences in the variable domains of their antibody chains.This serves to increase the diversity of the antibody pool and impacts the antibody’s antigen-binding affinity.[35] Some point mutations will result in the production of antibodies that have a weaker interaction (low affinity) with their antigen than the original antibody, and some mutations will generate antibodies with a stronger interaction (high affinity).[36] B cells that express high affinity antibodies on their surface will receive a strong survival signal during interactions with other cells, whereas those with low affinity antibodies will not, and will die byapoptosis.[36] Thus, B cells expressing antibodies with a higher affinity for the antigen will outcompete those with weaker affinities for function and survival. The process of generating antibodies with increased binding affinities is called affinity maturation. Affinity maturation occurs in mature B cells after V(D)J recombination, and is dependent on help from helper T cells.[37]IgG structure and function. IgG molecules are tetramers of ∼150 kDa, which comprise a pair of identical heavy and light chains linked by disulphide bonds (green bars). The heavy chains contain a variable (V) domain (theVH domain; green) and three constant (C) domains, the CH1 domain, CH2 domain and CH3 domain (purple). By contrast, the light chains contain a V domain (the VL domain; orange) and a single C domain (the CL domain; purple). Highly selective binding of antigen (light blue) is a common hallmark of antibodies. This is mainly mediated by six loops, which are known as the complementarity-determining regions (CDRs), three of which are present in each of the VH and VL domains. The interaction of antibody with antigen is exemplified in this figure by the Fab of trastuzumab (Herceptin; Genentech, Inc. and F.Hoffman-LaRoche Ltd) in complex with ERBB2 (REF. 144). For clarity, only the V domains of trastuzumab anddomain IV of ERBB2 are shown (see inset). The remaining portion of the V domains, which are known as framework regions, function mainly as a structural scaffold to support the CDRs.Human IgG bound to an antigen on a target cell, particularly IgG1 and IgG3, can subsequently interact through its Fc region with IgG receptors (FcγRs) expressed by effector cellsor with complement component 1q (C1q), potentially supporting the destruction of target cells through antibody- dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), respectively. In addition, the Fc region of IgG can bind the salvage receptor FcRn after fluid-phase uptake by vascular endothelial cells and other cells, an interaction that contributes to the long (∼21 day) half-life of human IgG. Modifying the amino-acid sequence of IgG to tailor the interaction with one or more binding partners is a promising strategy to improve the clinical potentialof antibodies (TABLE 2). Effector functions (that is, ADCC and CDC) require the presence of the Fc-region glycan (dark blue) and are crucially influenced by its structure. The N-linked glycan that is attached to the conserved asparagine(Asn) residue at position 297 comprises a core structure of N-acetylglucosamineand mannose, plus additional carbohydrate residues, which can vary, including fucose, galactose, sialic acid and bisecting N-acetylglucosamine.The image in the upper left inset is reproduced, with permission, from Nature REF.144© Macmillan Publishers Ltd.
  • Potential targets for antibody therapy of cancer. (a–d) MAbs can be used to target a number of cancer-associated targets, including tumor-associated blood vessels (a), vascular growth factors (for example, VEGF) (b), diffuse malignant cells (for example, leukemia) (c), tumor cells within a solid tumor (d) and tumor- associated stroma (for example, fibroblasts) (e).
  • Examples of antibody-mediated signaling inhibition. (a) Binding of ligand to a growth factor receptor triggers a dimerization event and activationof a signaling cascade, leading to cellular proliferation and resistance to cytotoxic agents (a). (b,c) MAb-based signaling inhibition can occur byblocking the dimerization event (b) or by interfering with ligand binding (c).
  • CD19 monoclonal antibodies deplete B cells in vivo through macrophage mediated antibody dependant cellular cytoxicity in animal models and in vitro. CD19 monoclonal antibodies bind to cd19 on the b cell surface, which allow monocyte recognition through Fc gamma receptors and the initiation of b-cell killing and or phagocytosis by activated macrophages. Therapeutic monoclonal antibodies the IgG1 isotype, which effectively mediates Fc domain–based functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement fixation.Antibody-dependent cellular cytotoxicity. ADCC occurs when antibodies bind to antigens on tumor cells and the antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector cells.For example, neutrophils commonly express human FcγRI (CD64), FcγRII (CD32) and the B (lipid-anchored) isoform of FcγRIII (CD16). In contrast, human natural killer (NK) cells express only the A (transmembrane) isoform of CD16.These data suggest that Fc-receptor interactions underlie anti-tumor efficacy in mice and may be important for the activity of selected mAbsin the clinic. The required effector cell populations have not been defined but are presumed to include mononuclear phagocytes and/or NK cells.Manipulations of Fc-domain structure can customize antibody clearance and the interaction of Fc domains with cellular Fc receptors25–27.There is little published evidence that ADCC contributes to clinical responses. However, several groups have recently shown that the efficacyof rituximab in lymphoma is substantially greater in patients with ‘high responder’ Fc-receptor polymorphisms (e.g., amino acid 158 valine/valineas opposed to valine/phenylalanine or phenylalanine/phenylalanine)28,29. These findings indicate that interactions between the antibody Fc domainand the Fc receptor underlie at least some of the clinical benefit of rituximab, simply the importance of ADCC. However, there have been nosystematic demonstrations that effective unconjugated mAb therapy of human cancer induces abundant tumor infiltration by host leukocytes,and there are few if any such demonstrations even in optimized animal models. Therapy with an ADCC-inducing bispecific antibody can induceboth anti-HER2/neu antibodies and T-cell responses (H. Borghaei et al., Fox Chase Cancer Center, unpublished data). Accordingly, ADCC can beviewed as a mechanism to directly induce a variable degree of immediate tumor destruction that leads to antigen presentation and the inductionof tumor-directed T-cell responses
  • Figure 2 Complement-directed cytotoxicity. (a–d) Binding of mAbs to antigen on the cell surface(a) exposes binding site on mAbs for proteins that initiate the complement cascade (b), ultimately triggering the release of chemotactic factors (c) and the formation of the membrane attack complex (d), which promotes target-cell lysis. Initial studies showing procytotoxic, recent study showing inhibitory, most work done on rituximab (anti cd20)
  • Figure 1 ADCC-mediated adaptive immunity switch. MAbs bind to antigen on the tumor cell surface, providing the target for Fc receptors on the surfaceof natural killer (NK) cells. Cross-linking of receptors triggers release of perforin and granzymes that lyse the tumor cell (a). (b–d) Cell debris is taken up byantigen-presenting cells (b), which present the tumor antigens to B cells, triggering the release of antibodies with specificities for numerous epitopes on thetarget antigens (c) and cytotoxic T lymphocytes (CTLs) that are capable of recognizing and killing cells that express the target antigen (d).
  • One year following rituximab treatment, lymphoma patients ho- mozygous for FCGR3A-158V, encoding the high-affinity FcγRIIIA, had a 90% objective response rate, compared with a 67% objec- tive response rate for patients carrying the low- affinity FCGR3A-158F polymorphism (35). Similarly, breast cancer patients homozygous for FCGR3A-158V had significantly higher objective response rates following trastuzumab treatment compared with patients carrying FCGR3A-158F (82% versus 40%), and this higher response rate was associated with significantly longer progression-free survival A smaller study examining cetuximab treatment in colorectal cancer found that patients with either of the high-affinity polymorphisms, FCGR3A-158V or FCGR3A-131R, had a median progression-free survival of 3.7 months compared with 1.1 months for patients who carried neither high-affinity polymorphismCartronG,DacheuxL,SallesG,Solal-CelignyP,BardosP,etal.2002.Therapeuticactivityofhumanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcγRIIIa gene. Blood 99:754–58 Musolino A, Naldi N, Bortesi B, Pezzuolo D, Capelletti M, et al. 2008. Immunoglobulin G frag- ment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. J. Clin. Oncol. 26:1789–96 37. ZhangW,GordonM,SchultheisAM,YangDY,NagashimaF,etal.2007.FCGR2AandFCGR3Apoly- 
morphisms associated with clinical outcome of epidermal growth factor receptor expressing metastatic 
colorectal cancer patients treated with single-agent cetuximab. J. Clin. Oncol. 25:3712–18
  • Representative antibody formats. The modular domain architecture of immunoglobulins has been exploited to create a growing range of alternative antibody formats that spans a molecular-weight range of at least 12–150 kDa and a valency (n) range from monomeric (n = 1), dimeric (n = 2) and trimeric (n = 3) to tetrameric (n = 4) and possibly higher12,16. For simplicity, all antibody formats are shown as being monospecific: that is, having one or more copies of identical antigen-binding sites. However, formats with a valency of two or more have also been used to create antibodies that have two or more (up to the valency of the format) distinct antigen-binding sites, which bind different antigens or different epitopes on the same antigen. The building block that is most frequently used to create novel antibody formats is the single-chain variable (V)-domain antibody fragment (scFv), which comprises V domains from the heavy and light chain (VH and VL domain) joined by a peptide linker of up to ∼15 amino-acid residues. Both the VL-domain–peptide-linker–VH-domain topology and the VH-domain–peptide-linker–VL-domain topology have been widely used. Constant (C) and V domains, inter-chain disulphide bonds (green bars) and glycosylation are depicted as in FIG. 2, and peptide and chemical linkers are shown as orange and red lines, respectively. CH, C domain of immunoglobulin heavy chain; CL, C domain of immunoglobulin light chain;dsFv, disulphide-stabilized scFv.
  • Modified antibodies. For targeted administration agents can be fused via a linker to a complete antibody (most often humanized or human; a), to a bivalent antibody binding fragment F(ab)2 (b), to a single-chain variable fragment (scFv) (c) or to a minibody consisting of two scFv regions fused to heavy-chain constant region (CH)3 (d). Immunomodulatory antibodies can be targeted to the tumour by means of bispecificdiabodies containing two different scFV regions (e). CL, light-chain constant region; Fc, constant fragment of an antibody; VH, heavy-chain variable region; VL, light-chain variable region.
  • Internalization of antibody–drug conjugates. To regain their cytotoxic activity, the cytotoxic agent has to be cleaved from the chemo-immunoconjugate. Uptake of antibodies predominantly occurs via the clathrin-mediated endocytosis pathway. After binding the respective antigen associated with coated pits, antibody– drug conjugates will be readily endocytosed, from where they transit through several stages of transport and endosomal vesicles and finally end up in a lysosome. There, linkers and antibody will be cleaved releasing the cytotoxic agent which — after exit from the lysosomal compartment — exerts its cytotoxic effect. http://www.biooncology.com/research-education/adc/how-adcs-work/index.htmlAntibody-drug conjugate concept. An ADC comprises an antibody conjugated via a linker to a cytotoxic drug. ADCs are commonly in IgG format with 2–8 drugs/antibody. Some portion of an ADC delivered intravenously localizes to a tumor and binds to a target antigen on the surface of the tumor cell. Internalization of the ADC commonly proceeds via a clathrin-coated pit mechanism167 although other mechanisms such as calveolae168 and pinocytosis sometimes occur.32 Inter- nalized vesicles are stripped of clathrin, fuse with other vesicles and enter the endosome-lysosome pathway. Proteases in the acidic environment of the endosomes digest the antibody, and potentially also the linker, to release free drug. The drug then crosses the membrane to enter the cytoplasm where it binds to its molecular target, commonly leading to cell cycle arrest and apoptosis. A portion of the drug is effluxed from the cell, either by passive diffusion, active transport, or leakage from dy- ing cells. If the effluxed drug is cell permeable it may enter neighboring cells and give rise to so-called bystander cell kill- ing.25,131 In some cases, the drug is metabolized in the cell and these metabolites may differ from the parent drug in their ability to kill the tumor cells as well as in the propensity to be effluxed
  • Roche said patients treated with T-DM1 in a Phase II trial lived significantly longer without their breast tumors getting worse and suffered far fewer serious side effects than patients who received the standard combo of Roche's Herceptin and the chemo drug Taxotere. These data added creditability to the underlying technology of the drug, an "armed antibody," which consists of the HER2-targeting antibody Herceptin that attached with a chemical linker to the chemotherapy DM1. This approach could limit damage to non-cancerous tissues observed when chemo drugs are given as stand-alone agents.
  • Antibody-directed enzyme prodrug therapy (ADEPT). (a) The mAb- enzyme conjugate binds to tumor cell-surface antigen. (b–d) After unbound mAb-enzyme is actively or passively cleared from circulation, the cytotoxic agent is administered in an inactive (prodrug) form (b), which is selectively bound by the mAb-enzyme conjugate on the tumor cell surface (c), cleaving the inactivating sequence from the prodrug and releasing multiple copies of active drug in the tumor microenvironment A phase I study of single administration of antibody-directedenzymeprodrugtherapy with the recombinant anti-carcinoembryonic antigen antibody-enzyme fusion protein MFECP1 and a bis-iodo phenol mustard prodrug.Antibody enzyme fusion proteins for cancer therapy 3011, immunotherapy
  • Boost immunity against minimal residual disease, and prevent outgrowth of metastasis. B. Tumor specific t cells migrate to sites of tumor metastases where they attempt to kill tumor cells express antigens coainted in the vaccine. The function is compromised by the immunosuppressive tumor microenvironment.
  • A. Prophylactic before occurrence of tumors. Individuals at high risk, or premalignant disease. Antigens expected to be expresssed by the tumor. B. If tumor begins to grow, memory cells activated. C. Tumor not allowed to grow large and heterogeneous and is easily eliminated.
  • Approaches to antitumor vaccination. (A) Irradiated tumor cells transduced with a viral gene transfer vector encoding a cytokine such as GM-CSF attract APCs (DCs) that acquire, process, and present tumor-associated antigens (TAAs) encoded by the vector in the context of MHC. (B) DCs can be directly loaded by incubation with tumor protein lysates or peptides with sequences based on expressed tumor antigens, or by viral gene transfer vectors expressing TAAs. (C) TAAs can be locally supplied to DCs by the direct injection of peptides, viral gene expression vectors, or naked DNA expression plasmids. DCs migrate to secondary lymphoid tissues where they present the antigen epitopes to T cells to generate an antitumor cytolytic T cell response.
  • Phase 3 study
  • Progression-free survival vaccine was 2.2 compared with 1.6 Overall survival patients who received the vaccine was 17.8 monthscompared with 11.1 months among patients who did not receive the vaccine. (NOT SS)
  • Red blood cells and granulocytes are XXX from the apheresis product by, retaining the leukocytes. The GM-CSF portion of the protein helps to target the PAP protein to antigen presenting cells and activate those cells. The PAP provides the tumor specific antigen that will direct the immune system to target prostatecancer. The cells are cultured in the presence of PA2024 for 36-44 hours. After culture, the cells are washed and suspended in lactated ringer’s solution for infusion back into the patient. Potency of infused cells measured by CD54. CD54 used as surrogate n CD54 as a measure for potency and as a marker for the active component of the therapy. CD54 is not considered an APC marker and CD54 upregulation is not specific to monocytes. Nevertheless, Dendreon has demonstrated that the biological activity is present in the CD54+ fraction, and that upregulation of CD54 on the monocyte-rich population correlates with improved survival in the treatment arm.
  • Study 1 was a randomized, double-blind, placebo-controlled, multicenter trial in patients with asymptomatic or minimally symptomatic metastatic castrate resistant (hormone refractory) prostate cancer. Eligible patients had metastatic disease in the soft tissue and/or bone with evidence of progression either at these sites or by serial Prostate Specific Antigen (PSA) measurements. Exclusion criteria included visceral (liver, lung, or brain) metastases, moderate to severe prostate cancer-related pain, and use of narcotics for cancer-related pain. A total of 512 patients were randomized in a 2:1 ratio to receive PROVENGE (n=341) or control (n=171). The median age was 71, and 90% of the patients were Caucasian. Thirty-five percent of patients had undergone radical prostatectomy, 54% had received local radiotherapy, and 82% had received combined androgen blockade. All patients had baseline testosterone levels < 50 ng/mL. Forty-eight percent of patients were receiving bisphosphonates and 18% had received prior chemotherapy, including docetaxel. Eighty-two percent of patients had an ECOG performance status of 0; 58% had primary Gleason scores of four or more; 44% had bone and soft tissue disease; 48% had bone-only disease; 7% had soft tissue-only disease; and 43% had greater than ten bony metastases. Study 2 was a randomized, double-blind, placebo-controlled, multicenter trial in patients with metastatic castrate resistant prostate cancer and no cancer-related pain. The primary endpoint was time to disease progression; analysis of the primary endpoint did not reach statistical significance. All patients were to be followed for survival; however, the survival analysis was not pre-specified. A third study, similar in design to Study 2, was terminated prior to completion of planned accrual.
  • Current clinical protocols for adoptive cell therapy. Adoptive cell therapy (ACT) requires the generation of highly avid tumour-antigen-reactive T cells. Tumour- specific T cells, derived from tumour-infiltrating lymphocytes (TILs), can be efficiently isolated ex vivo from melanoma lesions using high levels of interleukin-2 (IL-2). TILs are successively selected for their ability to secrete high levels of interferon-γ (IFNγ) when cultured with autologous or allogeneic MHC-matched tumour-cell lines. Alternatively, cell-mediated lysis has been used to identify tumour-reactive T cells for transfer. Highly avid, tumour-antigen-reactive T-cell populations selected for ACT are rapidly expanded (to up to 10 to the 11th cells) using CD3-specific antibody, exogenously supplied IL-2 and irradiated allogeneic peripheral-blood mononuclear ‘feeder’ cells, and are validated for activity before transfer. Patients now receive systemic immunosuppression before the adoptive transfer of antitumour lymphocytes. Published lymphodepleting regimens consist of a non-myeloablative, but lymphodepleting, conditioning chemotherapy comprised of cyclophosphamide and fludarabinebefore administration of T cells. Newer, as yet unpublished, regimens also include total body irradiation. ELISA, enzyme-linked immunosorbent assay. In the latest Surgery Branch, national Cancer Institute trials multiple TIL cultures were initiated from freshly resected metastatic melanomas (a mass of ≥1cm3 was required) and as soon as anti-tumour activity could be detected in vitro against specific unique or shared antigens, cells underwent a rapid expansion using the T-cell-stimulating antibody OKT3 and IL2 (fIG. 1). Approximately 5×1010 cells were infused immediately following a non-myeloablative preparative regimen consisting of 60 mg/kg cyclophosphamide for 2 days followed by 5 days of fludarabine at 25 mg/m2. Twenty-five patients each also received either 2 Gy or 12 Gy plus the chemotherapy regimen. IL2 was administered for 2–3 days at 7.2×105 IU/kg every 8 h. In vitro Utilizing overnight cytokine release coculture assays against either autologous tumor- or human leukocyte antigen (HLA)-matched melanoma cell lines. Cultures with evidence of specific reactivity compared with allogeneic non-MHC–matched controls that exceeded 200 pg/mL of IFNg and were at least twice control values were selected for rapid expansion as previously described
  • Blood smears obtained from patient9(left) and patient 10(right) during the lymphocytic episodes showed the highly activated phenotype of the lymphocytes, which show irregular and hyperchromatic nuclei, a high nuclear-to-cytoplasmic ratio, toxic granulation, and the presence of Dohle bodies.
  • A tumor lesion excised from patient 9 before nonmyeloablating chemotherapy (“pretreat- ment”) exhibited scant CD8􏰇 cells (left), strong stromal cell staining but weak staining of tumor cells with antibody to MHC class I (center), and sporadic cell staining with an antibody to MHC class II ( probably tumor macrophages) but minimal staining of tumor cells (right). In contrast, a sample resected 57 days after cell transfer (“post treatment”) exhibited a dense, diffuse CD8􏰇 infiltrate and ubiquitous expression of both MHC class I and class II molecules in tumor cells. Increase in MHC attributed to cyotkine release by transferred lymphocytes which have infiltrated the tumor.
  • Patient response to treatment was assessed utilizing Response Evaluation Criteria in Solid Tumors (RECIST) guidelines starting at approximately 4 weeks after cell administration and at regular intervals thereafter. Complete Response (CR): Disappearance of all target lesionsPartial Response (PR): At least a 30% decrease in the sum of the LD of target lesions, taking as reference the baseline sum LDStable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment startedProgressive Disease (PD): At least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesionsThere was no significant difference in overall response rate among the cohorts.though there was an increased rate of complete responses associated with increasing dose of TBI (P2 1⁄4 0.007; Table 2; results are descriptive and not corrected for multiple comparisons). Cohorts accrued sequentially (interpreted with caution)For the 20 complete responders- 3 and 5 year survival was 100% and 93%. (18/20 received only one treatment, 2 received two treatments)For the 32 partial responders 31% and 21%, For the 41 nonre- sponders 7% and 5%, respectively.
  • Metastatic melanoma typically has a 5 year survival rate of 5%.For the 20 complete responders, the 3- and 5-year survival rates were 100% and 93%, For the 32 partial responders 31% and 21%, For the 41 nonre- sponders 7% and 5%, respectively.
  • T-cell specificity is mediated through the T-cell antigen receptor (TCR). The TCR consists of α- and β-chains that genetically determine the antigen specificity of T cells in a clonotypic fashion. The α/β heterodimer recognizes peptides presented by major histocompatibility complex (MHC)-encoded molecules that are referred to as human leukocyte antigens (HLA). Antigen recognition is therefore restricted by each individual’s HLA background. Whereas antigen specificity is imparted through the of α- and β-chains, signalling is mediated through the CD3 complex of the TCR, which consists of a δ−ε-chain heterodimer, a γ−ε-chain heterodimer and a ζ-chain homodimer. Following binding of the HLA-peptide complex, the immunoreceptor tyrosine-based activation motifs (ITAM) (rectangles), which are present in the chains of the CD3 complex, become phosphorylated, initiating the process of T-cell activation115. The diagram also shows the B-cell antigen receptor (BCR, left), in which antigen recognition is mediated by a transmembraneimmunoglobin (Ig) and activation by the Igα and Igβ co-receptors. The Ig is a dimer of a heavy (H) and light (L) chain, each of which consists of variable (V; green) and constant (C; brown) regions. The antigen specificity of the receptor FcεR1 (right), which is expressed by natural-killer cells, is imparted by α- and β-chains, and activation is mediated by the FcRγ dimer. The Igα, Igβ, CD3ε and FcRγ each contain one ITAM motif, whereas CD3ζ contains three.
  • Principles of CAR architecture. CARs consist of a binding moiety, an extracellular hinge and spacer element, a transmembrane region (TM), and the signaling endodomain. Commonly the binding moiety consists of scFv derived from a TAA-specific monoclonal antibody and the signaling domain(s) come(s) from activating and costimulatory immune receptors.
  • Evolution of CAR signaling capacities. First generation CARs transmitted activating signals only via ITAM-bearing signaling chains like CD3ζ or FcεRIγ, licensing the engrafted T cells to eliminate tumor cells. Second generation CARs contain an additional costimulatory domain (CM I), predominantly the CD28 domain. Signaling through these costimulatory domain leads to enhanced proliferation, cytokine secretion, and renders engrafted T cells resistant to immunosuppression and induction of AICD. Recent developments fused the intracellular part of a second costimulatory molecule (CM II) in addition to CD28 and ITAM-bearing signaling chains, thus generating tripartite signaling CARs. T cells engrafted with third generation CARs seem to have superior qualities regarding effector functions and in vivo persistence.
  • Antitumor effects mediated by CAR-engrafted T cells. CAR-modified T cells can recognize tumor cells via binding of the CAR to its TAA independent of TCR-MHC/Peptide interactions. As a result T cells are activated and can efficiently eliminate tumor cells by the secretion of perforin and granzymes as well as the expression of FasL and tumor necrosis factor-related apoptosis inducing ligand (TRAIL). In addition, other tumor-infiltrating immune cells can be activated by the secretion of various cytokines. A major advantage of endowing T cells with non-MHC-restricted, antibody-derived specificity is that the potential target structures are no longer restricted to protein-derived peptides, but rather comprise every surface molecule on tumor cells including proteins with varying glycosylation patterns and nonprotein structures like gangliosides or carbohydrate antigens
  • gene delivery vehicles (vectors) thanks to their ability to integrate into the genome of non-dividing cells, which is the unique feature of Lentiviruses as other Retroviruses can infect only dividing cells.Lentivirus uses intergrase to intergrate DNA into genome. (intergrase has lower tendancy to insert in places causing oncogenesis) Replication competent (1-3) Random
  • Antileukemic effects in patient 1 and 3. Patient 2 had clinically documented disease after steroid admin.
  • Panel C shows bone marrow–biopsy specimens ob- tained 3 days after chemotherapy (day –1, before CART19-cell infusion) and 23 days and 6 months after CART19-cell infusion (hematoxylin and eosin). The baseline specimen shows hypercellular bone marrow (60%) with trilineage hematopoiesis, infiltrated by pre- dominantly interstitial aggregates of small, mature lymphocytes that account for 40% of total cellularity. The specimen obtained on day 23 shows residual lym- phoid aggregates (10%) that were negative for chronic lymphoid leukemia (CLL), with a mixture of T cells and CD5-negative B cells. The specimen obtained 6 months after infusion shows trilineage hematopoiesis, without lymphoid aggregates and continued absence of CLL.
  • Panel D shows contrast-enhanced CT scans obtained before the patient was enrolled in the study and 31 days and 104 days after the first infusion. The preinfusion CT scan reveals 1-to-3-cm bilateral masses. Regression of axillary lymphadenopathy occurred within 1 month after infusion and was sustained. Arrows highlight vari- ous enlarged lymph nodes before therapy and lymph- node responses on comparable CT scans after therapy.
  • Sequential BM biopsy from patient one were stained for CD20. Normal cellularity and trilineagehematopoesis observed.
  • Antileukemic effects in patient 1 and 3. Patient 2 had clinically documented disease after steroid admin.
  • Genomic DNA (gDNA) was isolated from samples of the patient’s whole blood (Panel A) and bone marrow aspirates (Panel B) collected at serial time points before and after chimeric antigen receptor T-cell infusion and used for quantitative real-time polymerase-chain-reaction (PCR) analysis. As assessed on the basis of transgenic DNA and the percentage of lymphocytes expressing CAR19, the chimeric antigen receptor T cells expanded to levels that were more than 1000 times as high as initial engraftment levels in the peripheral blood and bone marrow. Peak levels of chimeric antigen receptor T cells were temporally correlated with the tumor lysis syndrome. A blood sample ob- tained on day 0 and a bone marrow sample obtained on day −1 had no PCR signal at baseline. Flow-cytometric analysis of bone marrow aspirates at baseline

Basics of Immunotherapy Basics of Immunotherapy Presentation Transcript

  • Current concepts in cancerimmunotherapeuticsChiraag D. Patel, MDPGY2Stonybrook University Medical Center
  • Objectives• Background• Brief overview of immunology• Antibody based therapy• Vaccine based therapeutics• Adoptive cell therapy
  • Background • Bone surgeon at New York Cancer Hospital (Now MSKCC) • While going through hospital records, Coley found a sarcoma case study of one patient named Fred Stein, whose tumor disappeared following a high fever from Strep Pyogenes (Erysipelas) • Treated patients by injecting brew of strep pyogenes directly into inoperable tumors. • First patient treated in 1893 with Coley vaccineSir William Coley • 16 year old boy with abdominal tumor • Clinical presentation of infectious disease with fever, chills, gradual remission of tumor over 4 months of treatment • Patient lived another 26 years, died of myocardial infarction
  • Evidence for tumor immunity• spontaneous regression• regression of metastases after removal of primary tumor• infiltrations of tumors by lymphocytes and macrophages• lymphocyte proliferation in draining lymph nodes• increased cancer risk after immunosuppression and immunodeficiency
  • Overview of immune response
  • MHC-Major Histocompatibility Complex: Role of MHC Molecules• Membrane-bound glycoproteins- Class I and Class II MHC molecules- MHC genes highly polymorphic-within eachspecies there are manydifferent forms (alleles)• MHC function as antigen-recognitionmolecules- Can bind to a spectrum of antigenic peptides;polymorphism allowsfor diversity in antigen recognition• MHC I ---expressed on all nucleated cells - Present endogenous antigens-CD 8 + (Tc cells) -recognize antigen on MHC I• MHC II ---expressed on Antigen presentingcells (APC)- Present exogenous antigen- CD 4 + (Th cells) -recognize antigen on MHC II
  • How mhc works
  • Professional antigen presenting cells
  • Tumor antigens
  • Anti-tumour immunity: basic mechanisms •Presence of tumour cells and tumour antigens may initiate the release of “danger” signals; cytokines, heat shock proteins (HSP), uric acid etc. • Activation and maturation of dendritic cells, which present tumour antigens to CD8 and CD4 cells • Clonal expansion of CD8 and CD4 T cells; migration from the lymph node • Subsequent T cell mediated destruction of tumour cells Smyth, M. J. et al. Nature Immunology 2, 293 - 299 (2001)
  • ANTIBODY BASED THERAPY
  • Immunoglobulin structure
  • Targets
  • Direct Mechanism Nature biotechnology, Weiner, 2005
  • Short term Mechanism Nature Reviews Rheumatology, Tedder, 2009
  • Complement-directed cytotoxicity Nature biotechnology, Weiner, 2005
  • Long term mechanism Nature biotechnology, Weiner, 2005
  • Affinity of FcγRIIIa receptor Drug measurement High Affinity Low Affinity receptor receptor Rituximab Objective 90% 67% Cd20 response Trastuzumab Objective 82% 40% Her2/neu response cetuximab Median 3.7 month 1.1 month epidermal progression growth factor free survivalTherapeutic activity of humanized anti-CD20 monoclonal antibody (rituximab) and polymorphism in IgG Fc receptor FcγRIIIa gene.Blood 99:754–58Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. J. Clin. Oncol. 26:1789–96FCGR2AandFCGR3A polymorphisms associated with clinical outcome of epidermal growth factor (cetuximab ) receptorexpressing metastatic colorectal cancer patients treated with single-agent cetuximab. J. Clin. Oncol. 25:3712–18
  • Future concepts Nature Review Immunology, Carter, 2006
  • Antibody drug conjugate
  • Antibody drug conjugates
  • Nature biotechnology, Senter, 2005
  • Selected immuno conjugates Name Drug Target Cancer Trastuzumab DM1 emtansine Her2 breast (mertansine) Cervical head and neck Pseudomonas lung cancer SS1(dsFv)-PE38 Mesothelin exotoxin mesothelioma ovarian pancreatic cancer 5T4 Renal clear cell Staphylococcal ABR-217620 (Trophoblast NSCLC enterotoxin E glycoprotein) pancreatic Colorectal90Y-Labetuzumab 90Y CEA Breast pancreatic
  • ADEPT Nature biotechnology, Senter, 2005 Nature biotechnology, Weiner, 2005
  • VACCINE BASED THERAPY
  • Shifting the balance Journal of Boimedicine and Biotechnology, Schlom, 2010
  • Vaccines can drive clonal expansion of self-reactive T cells that escape negative selection Tumor cells Vaccine- bearing self-Ag Negative Driven Selection Clonal Self-Ag Expansion Development in thymus
  • Therapeutic vaccine Nature reviews Immunology, Finn, 2005
  • Prophylactic vaccine Nature reviews Immunology, Finn, 2005
  • Types of vaccines Journal of Biomedicine, Biotechnology, Tasng, 2010
  • Journal of Clinical Investigation, Berzofsky, 2004
  • Journal of Clinical Investigation, Berzofsky, 2004
  • Peptide vaccineGP100- MELANOMA
  • Gp100 + Il2• Randomized, phase 3 trial – 185 patients at 21 centers. – stage IV or locally advanced stage III cutaneous melanoma• Expression of HLA*A0201, an absence of brain metastases, and suitability for high dose interleukin-2 therapy.• Patients were randomly assigned to receive – interleukin-2 alone – interleukin-2 + gp100:209-217(210M) +incomplete Freund’s adjuvant (Montanide ISA-51)• The primary end point was clinical response.• Secondary end points included toxic effects and progression-free survival. NEJM, Hwu, 2011
  • Peptide vaccine • Peptide of gp100 (hmb45) plus incomplete Freund’s adjuvant (Montanide ISA-51) • gp100:209-217(210M) IMDQVPFSV • Montanide - Comprises an injectable pharmaceutical mineral oil and an extremely refined emulsifier obtained from mannitol and purified oleic acid of vegetable origin.
  • NEJM, Hwu, 2011
  • Dendritic cell vaccineAPC8015- PROSTATE
  • APC8015 mechanism ≥ 50 x 10 to the 6th CD54+ cell
  • Results
  • Clinical and Vaccine Immunology, Buonaguro, 2011
  • Clinical and Vaccine Immunology, Buonaguro, 2011
  • ADOPTIVE CELLULARIMMUNOTHERAPY
  • • What is Adoptive Immunotherapy: – Autologous or allogeneic WBC are coupled with growth factor(s) to enhance their cancer-fighting capacity - (+/-) specific antigen – Then, these are reinfused to increase immune response locally
  • Types of ACI• Autologous T cell transfer• Genetically modified t cell transfer (CAR)
  • Nature reviews immunology, Restifo, 2006
  • Trial• Ninety-three patients with measurable metastatic melanoma – Metastatic lesion >2cm – 86% stage M1b or M1c – 83% refractory to prior treatment• Treated with transfer of autologous TILs + interleukin-2 following lymphodepleting preparative regimen on three sequential clinical trials – Either chemotherapy, 2GY TBI, or 12 GY TBI• 95% of patients had progressive disease, and prior systemic treatment.• Median potential follow-up was 62 months. Clinical Cancer Research, Rosenberg, 2011
  • Type of response Science, Rosenberg, 2002
  • Type of responseMART-1= Melan Agp100= HMB 45 Science, Rosenberg, 2002
  • Results Science, Rosenberg, 2002
  • Science, Rosenberg, 2002
  • Clinical Cancer Research, Rosenberg, 2011
  • Clinical Cancer Research, Rosenberg, 2011
  • Results of TrialPR partial response SurvivalCR complete response 3 year 5 yearOR objective response CR(20) 100% 93% PR(32) 31% 21% NR(41) 7% 5% Clinical Cancer Research, Rosenberg, 2011
  • Long term results Clinical Cancer Research, Rosenberg, 2011
  • GENETICALLY MODIFIED T CELLTHERAPY (CHIMERIC ANTIGENRECEPTOR)
  • Nature Clinical Oncology, Rosenberg, 2011
  • Nature reviews cancer, Brentjens, 2003
  • Journal of Biomedicine and Biotechnology, Schmitz, 2010
  • 4-1BBJournal of Biomedicine and Biotechnology, Schmitz, 2010
  • Journal of Biomedicine and Biotechnology, Schmitz, 2010
  • Manufacturing
  • CART19 cell therapy study• Three patients with advanced, chemotherapy-resistant CLL were enrolled in a pilot clinical trial for CART19 cell therapy. Figure 1 presents a summary of the manufacturing process for the gene-modified T cells (A) and the clinical protocol design (B). All patients were extensively pretreated with various chemotherapy and biologic regimens (Table 1). Two of the patients had p53-deficient CLL, a deletion that portends poor response to conventional therapy and rapid progression (10). Each of the patients had a large tumor burden after the preparative chemotherapy, including extensive BM infiltration (40 to 95%) and lymphadenopathy; UPN 02 also had peripheral lymphocytosis. There was a low abundance of T cells in the apheresis products (2.29 to 4.46%) (table S1) as well as likely impaired T cell activation, as has been shown previously in CLL patients (11). Additional details of the cell manufac- turing and product characterization for the CART19 cell preparation for each patient are shown in table S1. All patients were pretreated 1 to 4 days before CART19 cell infusions with lymphodepleting chemo- therapy (Table 1). A split-dose cell infusion schedule was used to address potential safety concerns related to the evaluation of a previously untested CAR that incorporated the 4-1BB costimulatory signaling domain.
  • Patient 3 NEJM, Science, June/Kalos, 2011
  • Patient 3 NEJM, Science, June/Kalos, 2011
  • IHC CD20 NEJM, Science, June/Kalos, 2011
  • NEJM, Science, June/Kalos, 2011
  • Results x NEJM, Science, June/Kalos, 2011
  • Results• All patients displayed varying degrees of non infectious febrile illness/ tumor lysis syndrome NEJM, Science, June/Kalos, 2011
  • x Human Molecular Genetics, Heslop, 2011
  • CONCLUSION
  • Adverse effects of immunotherapy
  • Timing of immunotherapy
  • THE END
  • Responses to peptide-pulsed dendritic cellvaccine in melanoma patients :Enhanced T cell reactivity to melanoma antigenscorrelates with clinical response Overnight culture culture with peptide Banchereau et al, Cancer Res 61:6451 (2001)
  • CTLA-4 blockade powerfully enhances vaccine-mediated tumor immunity Treatment Day_______________ Days after SQ B16 tumor injection Hamster IgG B16/GMCSF + B16/GMCSF aCTLA-4 Hamser IgG +aCTLA-4 Therapy 1 day after iv injection of metatastic B16 Van Elsas et al, J.Exp.Med.190:355 (1999)
  • Areas of design• Improving selectivity for antigen• Antigen binding specificity, affinity • Improve efficacy • Improve ADCC• Effector function• Pharmacokinetics • Increase or reduce half life