Image_1_Tumor beta2-microglobulin and HLA-A expression is increased by immunotherapy and can predict response to CIT in association with other biomarkers.tif

BackgroundDownregulation of MHC class I expression and/or defects in the antigen presentation pathways are commonly reported in human cancers. Numerous studies previously have explored extensively the molecular mechanisms that underlie HLA-class I and Beta2-Microglobulin (B2M) downregulation. However, the techniques presently available to detect expression of MHC class I proteins lack the robustness, specificity and sensitivity needed for systematic integration and analysis in clinical trials. Furthermore, the dynamics of HLA-class I and B2M expression have not been comprehensively studied as a potential biomarker for immunotherapy.MethodsUsing novel, validated, immunohistochemistry (IHC)-based methods for quantifying B2M and HLA-A in tumor samples from diverse cancer types, we have determined loss of B2M and HLA-A proteins in 336 archived, primary specimens and 329 biopsies from metastatic patients collected during Roche-sponsored Phase 1 clinical trials investigating novel immunotherapy candidates as monotherapy or in combination with CPI.ResultsUp to 56% of cases with B2M or HLA-A loss were noted in the investigated tumor types. The frequency of loss was dependent on indication and stage of disease and revealed heterogeneous expression patterns across patients. B2M and HLA-A loss was increased in metastatic lesions compared to primary tumors, indicating selection of MHC class I low clones in metastatic and refractory tumor cells. High on-treatment B2M expression correlated with successful clinical outcome (RECIST), while high baseline B2M did not. A treatment-induced increase of B2M expression was noted in most of the patients with low B2M levels at baseline. The triple biomarker combination of B2M, CD8 and PDL1 strongly improved response prediction to cancer immunotherapy.ConclusionOur results indicate that B2M and HLA-A loss occurs frequently in tumors and is reversed in most instances following immunotherapy which supports the conclusion that MHC class I loss is not the dominant resistance mechanism to CPI treatment. This investigation reveals a highly dynamic expression of HLA-A and B2M in tumors affected by indication, metastatic status, immunophenotype and immunotherapy treatment. Baseline expression levels of B2M on tumors may be of utility as a constituent of a biomarker panel used for selecting patients for immunotherapy clinical trials.</p

ELISA titers against microtiter wells coated with synthetic Fba peptide.

<p>ELISA titers against microtiter wells coated with synthetic Fba peptide.</p

β-Man)₃-Fba-TT conjugate with or without adjuvant markedly induced high antibody titers and protection against disseminated candidiasis in immunized mice as compared to controls.

<p>Mice immunized with β-(Man)₃-Fba-TT prepared in either alum or MPL, or without adjuvant developed robust antibody responses against both the Fba peptide (A) and the β-(Man)₃ epitope (B). (C) Protective immunity was induced by (β-Man)₃-Fba-TT when either alum or MPL was used as the adjuvant. Protection was nearly as great even when adjuvant was omitted as compared to DPBS or adjuvant only controls (<i>P</i><0.01). (D) Immunized mice had reduced or non-detectable CFUs per kidney pairs compared to control groups (<i>P</i><0.001).</p

Antibody isotype distribution of responses to Fba and β-(Man)₃.

<p>Antibody isotype distribution of responses to Fba and β-(Man)₃.</p

Comparison of DC/CFA and alum as adjuvants for induction of immune resopnses to the β-(Man)₃-Fba conjugate vaccine.

<p>Serum samples were collected 14 days after immunization, diluted 1∶100 and tested by ELISA on plates coated with either synthetic Fba-MAP or β-(Man)₃. Immune sera from mice immunized with the β-(Man)₃-Fba DC/CFA showed greater antibody titers to both the Fba peptide (A) and the β-(Man)₃ epitopes (B) than sera from groups that received β-(Man)₃-Fba in alum. (C) A high degree of protection was induced by the β-(Man)₃-Fba pulsed DCs, and slight protection was observed when alum was used as the adjuvant as compared to DPBS, DC+CFA or alum adjuvant unimmunized controls.</p

ELISA titers against microtiter wells coated with synthetic β-(Man)₃ epitope.

<p>ELISA titers against microtiter wells coated with synthetic β-(Man)₃ epitope.</p

β-(Man)₃-Fba administered along with either alum or MPL adjuvants induced modest antibody responses and slight protection against disseminated candidiasis.

<p>The β-(Man)₃-Fba conjugate was administered as a mixture with either alum or MPL adjuvants in BALB/c mice. Serum samples were collected 14 days after immunization, diluted 1∶100 and tested by ELISA on plates coated with synthetic β-(Man)₃ or Fba-MAP. After the first booster immunization, immune sera from vaccinated mice showed modest antibody responses to Fba peptide (A) and relatively weak antibody responses to β-(Man)₃ epitope (B) (C) The survival was also slightly extended in mice that received β-(Man)₃-Fba in MPL and slight protection was observed when alum was used as the adjuvant as compared to DPBS or adjuvant unimmunized controls.</p

Passive transfer experiment was performed to confirm that antibody is responsible for the protection.

<p>(A) As immunized mice, immune sera recipients had a prolonged survival time (<i>P</i><0.01), confirming that induced antibodies were protective. (B) The serum from (β-Man)₃-Fba-TT immunized animals was capable of reducing the fungal load in the mouse kidneys compared with the infectious burden in mice were given DPBS or pre-absorbed sera (<i>P</i><0.001).</p

Vaccination with β-(Man)₃-Fba-TT in either alum or MPL markedly increased both β-(Man)₃ and Fba peptide-specific antibody titers in sensitized mice as compared to controls.

<p>Serum samples were collected 14 days after immunization, diluted 1∶100 and tested by ELISA on plates coated with cell wall mannan or peptide. MAbs B6.1 and E2-9 that are specific for β-(Man)₃ and Fba, respectively, were used as positive controls. Mice immunized with β-(Man)₃-Fba-TT prepared in either alum or MPL induced robust antibody responses against both the Fba peptide (A) and the β-(Man)₃ (B) epitopes. However, mice that received either Fba or Fba-TT in either adjuvant produced weak anti-Fba responses.</p

The (β-Man)₃-TT conjugate vaccine is immunogenic and protective against disseminated candidiasis in outbred mice.

<p>Outbred CFWSwiss Webster (01S60) mice were immunized with the β-(Man)₃-Fba-TT conjugate alone or mixed with adjuvants alum and MPL; control mice were immunized with adjuvants (alum+MPL) only or DPBS buffer. Mice immunized with β-(Man)₃-Fba-TT in either alum+MPL, or without adjuvant induced robust antibody responses against both the β-(Man)₃ epitope (A) and the Fba peptide (B). (C) Protective immunity was induced by (β-Man)₃-Fba-TT with or without adjuvant as noted by their prolonged survival time as compared to control mice that received DPBS or adjuvants alone (<i>P</i><0.01). (D) Immunized mice had reduced or non-detectable CFUs per kidney pairs compared to control groups.</p