Peptide Vaccines and Cancer Immunotherapy
Peptide Vaccines and Cancer Immunotherapy
In our continuing efforts to rationally design effective vaccines, we have taken advantage of information garnered from antigen–antibody complexes of HER-2 protein with clinically important antibodies. As such, we can design efficacious functional vaccines that mimic the 3D epitopes.
Numerous x-ray crystallographic structural studies revealing how the HER family initiates signal transduction have been published. The structure of soluble HER-2–trastuzumab Fab complex shows that the trastuzumab binding region is located on the C-terminus of the HER-2 ECD domain IV and this complex buries 1350 Å of the HER-2 surface with three loops residues 579–583, 615–625 and 592–595. On the other hand, the crystal structure of the HER-2 ECD bound to pertuzumab shows a different binding epitope focused on domain II residues 266–333. This structure provides a model in which pertuzumab sterically interferes with HER-2 dimerizing with other members of the HER family.
Our first-generation HER-2 peptide vaccines (316–339 and 628–647) were identified prior to the publications of the crystal structure of the HER-2 ECD using computer-aided analysis. The structures of the HER-2–trastuzumab and HER-2–pertuzumab complexes have led to new understandings of the mechanistic and biological activities of HER-2 antibodies as well as the process of ligand-induced receptor dimerization, which, in turn, has empowered us to rationally design more effective HER-2 conformational epitopes; the trastuzumab-binding epitope (597–626) and the pertuzumab-binding epitope (266–296).
The crystal structure of pertuzumab bound to the ECD of HER-2/neu revealed the details of interacting region of residues 266–333 (Figure 4). We designed and studied the important binding sequences spanning residues 266–296, 298–333 and 315–333 in order to define the most biologically relevant conformational epitope to mimic the pertuzumab-binding conformational region for effective vaccination (Table 2).
(Enlarge Image)
Figure 4.
Binding interface of pertuzumab and HER-2.
Important binding residues at the interface of pertuzumab and HER-2 show the key amino acid residues that are critical for binding. (I–III) are the HER-2 domains.
We reported on the extensive in vitro and in vivo results obtained by the various constructs having complex disulfide bonds, as well as the noncyclized (non-Cyc.) linear peptides. We vaccinated both mice and rabbits in order to elucidate the immunogenicity of the cyclized (Cyc.) and non-Cyc. constructs. We found that each of the epitopes was able to elicit high-affinity, high-titer antibody responses that bound the native HER-2/neu receptor. Additionally, only three of the six putative constructs – 266–296 (Cyc.), 266–296 (non-Cyc.) and 315–333 (Cyc.) – were able to reduce the phosphorylation of the HER-2/neu tyrosine kinase domain and to mediate ADCC. Additionally, we were able to show that epitope 266–296 was able to suppress cellular proliferation in heregulin-stimulated MCF-7 cells.
Our experiments in two transplantable tumor mice models (Balb/c and FVB/n) show that only the 266–296 engineered epitopes had statistically reduced tumor onset. We also demonstrated that there was significant reduction in tumor development in Balb-neuT and VEGFNeu2–5 transgenic mouse tumor models. In conclusion, we were able to show that the epitope spanning sequences 266–296 was by far the best candidate to elicit an efficacious antitumor immune response. This signifies that the 266–296 peptide vaccine could duplicate the antitumor effects of pertuzumab in vivo without the concomitant harmful side effects associated with mAb therapy.
The 3D structure of the complex (Figure 5) between human HER-2 and trastuzumab revealed that the region of HER-2 spanning residues 563–626 of the antigen-binding domain harbors a complex disulfide bonding pattern. In order to minimally dissect the interacting region of HER-2 binding domain, four synthetic peptides with different levels of structural flexibility were designed and synthesized. The interacting loops in subdomain IV comprise residues in loop 1: 579–583 (two disulfide pairings between C563 and C576, and between C567 and C584); loop 2: 592–595 (cysteine disulfide pairing between C587 and C596); and loop 3: 615–625 (cysteine disulfide between C600 and C623). The chimeric peptides in Table 3 were successfully synthesized, purified and characterized. All conformation-restricted peptides were able to bind to trastuzumab with 563–598 and 597–626 showing higher reactivity.
(Enlarge Image)
Figure 5.
Binding interface of trastuzumab to HER-2.
The crystal structure of HER-2 (red) in contact with trastuzumab (blue). The three loops that make direct contact with trastuzumab are clearly shown, depicting important binding residues.
All four peptide sequences inhibited tumor cell proliferation. Although all four sequences were immunogenic in FVB/N mice, only anti-597–626 and anti-613–626 were able to bind HER-2. We further examined the immunogenicity of the 597–626 epitope in outbred rabbits and high-titered antibodies were elicited that recognized HER-2 at the HER-2–trastuzumab interface, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and mediated ADCC. Antibodies against the 597–626 construct were able to mediate both direct and indirect mechanisms of antitumor activity against HER-2 in vitro.
We also demonstrated in transgenic BALB-neuT mice that immunization with the 597–626 epitope significantly reduced tumor burden. In conclusion, our results imply that the 597–626 peptide could be used as a vaccine for HER-2-overexpressing cancers since the resulting antibodies show similar biological activities to trastuzumab.
We are currently conducting an FDA-approved (investigational new drug 14633) and NCI-funded trial at the Ohio State University James Cancer Hospital (2010C0075; OSU 09138) entitled 'Phase I Active Immunotherapy Trial with a Combination of Two Chimeric HER-2 B Cell Peptide Vaccines emuslified in ISA 720 and nor-MDP in Patients with Advanced Solid Tumors' (NCT01376505). The combination peptide vaccine from the trastuzumab-like (MVF-HER-2 [597–626]) and pertuzumab-like (MVF-HER-2 [266–296]) binding sites have demonstrated efficacy in preclinical studies. The vaccine targets two different epitopes of the HER-2 ECD (II and IV), making it beneficial to both HER-2-positive and HER-2-negative (EGFR overexpressing) cancer patients. The dose-escalation trial was opened for accrual in July 2011; we have completed two cohorts and the trial is still open for accrual (cohorts 3 and 4).
The main objectives of this study will be to perform an early-phase clinical trial assessing safety and clinical toxicity of immunization with two HER-2 multivalent vaccines in patients with advanced solid tumors, as well as to establish a biologically optimum dose of combination vaccines with nor-MDP as adjuvant emulsified in ISA 720. We will also evaluate whether the combination of HER-2 epitopes shows therapeutic benefit, provides synergistic and/or additive effects and enumerate mechanisms of action. Once the biologically optimum dose has been established, the protocol calls for extending the trial in a Phase IIb efficacy trial in two indications. After completion of the Phase I trial (four cohorts, 24 patients), we will decide, based on evaluation of the results of the trial, to select two appropriate indications to extend the trial.
Developing the Second-generation HER-2 Vaccines
In our continuing efforts to rationally design effective vaccines, we have taken advantage of information garnered from antigen–antibody complexes of HER-2 protein with clinically important antibodies. As such, we can design efficacious functional vaccines that mimic the 3D epitopes.
Structural Studies of HER-2 With Pertuzumab & Trastuzumab
Numerous x-ray crystallographic structural studies revealing how the HER family initiates signal transduction have been published. The structure of soluble HER-2–trastuzumab Fab complex shows that the trastuzumab binding region is located on the C-terminus of the HER-2 ECD domain IV and this complex buries 1350 Å of the HER-2 surface with three loops residues 579–583, 615–625 and 592–595. On the other hand, the crystal structure of the HER-2 ECD bound to pertuzumab shows a different binding epitope focused on domain II residues 266–333. This structure provides a model in which pertuzumab sterically interferes with HER-2 dimerizing with other members of the HER family.
Our first-generation HER-2 peptide vaccines (316–339 and 628–647) were identified prior to the publications of the crystal structure of the HER-2 ECD using computer-aided analysis. The structures of the HER-2–trastuzumab and HER-2–pertuzumab complexes have led to new understandings of the mechanistic and biological activities of HER-2 antibodies as well as the process of ligand-induced receptor dimerization, which, in turn, has empowered us to rationally design more effective HER-2 conformational epitopes; the trastuzumab-binding epitope (597–626) and the pertuzumab-binding epitope (266–296).
Design & Evaluation of Novel Pertuzumab-binding Conformational B-cell Epitopes
The crystal structure of pertuzumab bound to the ECD of HER-2/neu revealed the details of interacting region of residues 266–333 (Figure 4). We designed and studied the important binding sequences spanning residues 266–296, 298–333 and 315–333 in order to define the most biologically relevant conformational epitope to mimic the pertuzumab-binding conformational region for effective vaccination (Table 2).
(Enlarge Image)
Figure 4.
Binding interface of pertuzumab and HER-2.
Important binding residues at the interface of pertuzumab and HER-2 show the key amino acid residues that are critical for binding. (I–III) are the HER-2 domains.
We reported on the extensive in vitro and in vivo results obtained by the various constructs having complex disulfide bonds, as well as the noncyclized (non-Cyc.) linear peptides. We vaccinated both mice and rabbits in order to elucidate the immunogenicity of the cyclized (Cyc.) and non-Cyc. constructs. We found that each of the epitopes was able to elicit high-affinity, high-titer antibody responses that bound the native HER-2/neu receptor. Additionally, only three of the six putative constructs – 266–296 (Cyc.), 266–296 (non-Cyc.) and 315–333 (Cyc.) – were able to reduce the phosphorylation of the HER-2/neu tyrosine kinase domain and to mediate ADCC. Additionally, we were able to show that epitope 266–296 was able to suppress cellular proliferation in heregulin-stimulated MCF-7 cells.
Our experiments in two transplantable tumor mice models (Balb/c and FVB/n) show that only the 266–296 engineered epitopes had statistically reduced tumor onset. We also demonstrated that there was significant reduction in tumor development in Balb-neuT and VEGFNeu2–5 transgenic mouse tumor models. In conclusion, we were able to show that the epitope spanning sequences 266–296 was by far the best candidate to elicit an efficacious antitumor immune response. This signifies that the 266–296 peptide vaccine could duplicate the antitumor effects of pertuzumab in vivo without the concomitant harmful side effects associated with mAb therapy.
Design & Evaluation of Novel Trastuzumab-binding Conformational B-cell Epitopes
The 3D structure of the complex (Figure 5) between human HER-2 and trastuzumab revealed that the region of HER-2 spanning residues 563–626 of the antigen-binding domain harbors a complex disulfide bonding pattern. In order to minimally dissect the interacting region of HER-2 binding domain, four synthetic peptides with different levels of structural flexibility were designed and synthesized. The interacting loops in subdomain IV comprise residues in loop 1: 579–583 (two disulfide pairings between C563 and C576, and between C567 and C584); loop 2: 592–595 (cysteine disulfide pairing between C587 and C596); and loop 3: 615–625 (cysteine disulfide between C600 and C623). The chimeric peptides in Table 3 were successfully synthesized, purified and characterized. All conformation-restricted peptides were able to bind to trastuzumab with 563–598 and 597–626 showing higher reactivity.
(Enlarge Image)
Figure 5.
Binding interface of trastuzumab to HER-2.
The crystal structure of HER-2 (red) in contact with trastuzumab (blue). The three loops that make direct contact with trastuzumab are clearly shown, depicting important binding residues.
All four peptide sequences inhibited tumor cell proliferation. Although all four sequences were immunogenic in FVB/N mice, only anti-597–626 and anti-613–626 were able to bind HER-2. We further examined the immunogenicity of the 597–626 epitope in outbred rabbits and high-titered antibodies were elicited that recognized HER-2 at the HER-2–trastuzumab interface, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and mediated ADCC. Antibodies against the 597–626 construct were able to mediate both direct and indirect mechanisms of antitumor activity against HER-2 in vitro.
We also demonstrated in transgenic BALB-neuT mice that immunization with the 597–626 epitope significantly reduced tumor burden. In conclusion, our results imply that the 597–626 peptide could be used as a vaccine for HER-2-overexpressing cancers since the resulting antibodies show similar biological activities to trastuzumab.
New Clinical Trial Peptides in the Clinic
We are currently conducting an FDA-approved (investigational new drug 14633) and NCI-funded trial at the Ohio State University James Cancer Hospital (2010C0075; OSU 09138) entitled 'Phase I Active Immunotherapy Trial with a Combination of Two Chimeric HER-2 B Cell Peptide Vaccines emuslified in ISA 720 and nor-MDP in Patients with Advanced Solid Tumors' (NCT01376505). The combination peptide vaccine from the trastuzumab-like (MVF-HER-2 [597–626]) and pertuzumab-like (MVF-HER-2 [266–296]) binding sites have demonstrated efficacy in preclinical studies. The vaccine targets two different epitopes of the HER-2 ECD (II and IV), making it beneficial to both HER-2-positive and HER-2-negative (EGFR overexpressing) cancer patients. The dose-escalation trial was opened for accrual in July 2011; we have completed two cohorts and the trial is still open for accrual (cohorts 3 and 4).
The main objectives of this study will be to perform an early-phase clinical trial assessing safety and clinical toxicity of immunization with two HER-2 multivalent vaccines in patients with advanced solid tumors, as well as to establish a biologically optimum dose of combination vaccines with nor-MDP as adjuvant emulsified in ISA 720. We will also evaluate whether the combination of HER-2 epitopes shows therapeutic benefit, provides synergistic and/or additive effects and enumerate mechanisms of action. Once the biologically optimum dose has been established, the protocol calls for extending the trial in a Phase IIb efficacy trial in two indications. After completion of the Phase I trial (four cohorts, 24 patients), we will decide, based on evaluation of the results of the trial, to select two appropriate indications to extend the trial.
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