In an article for npj vaccines in October 2024, researchers present their investigation into the efficacy of a combination of DNA vaccine encoding mouse GPRC5D and PD-1 in preventing and treating multiple myeloma (MM). MM “remains largely incurable”, but the GPRC5D, “highly expressed” in MM, presents a “compelling” immunotherapy candidate. The research suggests that GPRC5D-targeted DNA vaccines are “versatile platforms” for treating and preventing MM.
Managing MM
Multiple myeloma (MM) is the second most prevalent haematological malignancy, characterised by the accumulation of malignant plasma cells in bone marrow. Most MM cases are preceded by monoclonal gammopathy of undetermined significance (MGUS), which can reduce a person’s life expectancy by “more than 4 years”. Around 3.5 million people are affected by MGUS in the United States. Smouldering MM (SMM), distinguished from MGUS for clinical reasons, is an asymptomatic clonal plasma cell disorder between MGUS and MM.
MM treatment has been “transformed with the advent of antibody-based therapies”, with chimeric antigen receptor (CAR) T-cell therapies that target the B-cell maturation antigen (BCMA) showing “considerable promise”. However, the pattern of BCMA expression is heterogeneous, responsible for “varied treatment responses” and the surface expression can “fluctuate” because of gamma secretase-mediated shedding of the extracellular domain. Furthermore, antigen escape has been noted in patients with MM who experienced relapse after BCMA-targeted CAR T-cell therapy.
“Exploring immunotherapies targeting alternative antigens may help counteract antigen escape and provide effective treatment options for patients who relapse after BCMA-targeted CAR T-cell treatment.”
A new vaccine target
C protein-coupled receptors (GPCRs) are the “largest and most diverse” group fo membrane receptors in eukaryotes; humans have almost 1000 different GPCRs. GPCRs are classified into six classes (A-F), among which class C GPCRs initiate metabolic steps to modulate cellular activity.
Orphan GPCR class C group 5 member D (GPRC5D) is expressed in the hair follicle and the bone marrow of patients with MM, as well as in MGUS and SMM. The GPRC5D mRNA is overexpressed between two and four times in MM plasma cells compared to normal plasma cells, and immediate expression is seen in MGUS and SMM.
“GPRC5D is an emerging novel immunotherapeutic and preventive target for MM.”
Although DNA vaccines are a “promising” alternative to mRNA vaccines, with “lower cost and better stability”, they have not yet been widely adopted in clinical practice. DNA cancer vaccine development faces “significant challenges” such as nonspecific formulations, thermal instability, toxicity, and ineffectiveness. However, the authors believe that recent advancements have “greatly enhanced” the clinical efficacy of DNA vaccines in cancer treatment.
The study
In their research, the authors attempted to develop DNA vaccines against MM using plasmids expressing GPRC5D. First, they evaluated a mouse GPRC5D DNA vaccine in the 5TGM1 murine myeloma model, which “closely mimics” human MM. Cancer prevention activity was examined through administration of the DNA vaccine before tumour cell inoculation. The mice that received the mGPRC5D vaccine developed “significantly smaller” tumours than the control mice, and all animals in the mGPRC5D group were alive at day 33.
With ELISA, the authors evaluated the humoral response by measuring the levels of mGPRC5D-specific antibody in the serum collected 5 days after boost. They found a “marked increase” in serum IgG levels in the mGPRC5D group. To explore the possible mechanisms of the antitumour effect of the vaccine, they analysed immune cells in the spleen and tumours through flow cytometry. The percentage of various immune cell populations “significantly increased” in the mGPRC5D-immunised mice.
The research also considered the therapeutic efficacy of the mGPRC5D vaccine in combination with PD1 Ab treatment. After tumour inoculation, mice received two injections of 20µg mGPRC5D vaccine or the control plasmid at 2-week intervals, along with intraperitoneal administration of anti-PD1 antibody. Mice that received either the vaccine or anti-PD1 Ab showed a “moderate inhibitory effect”, but those treated with the combination exhibited “significant inhibition of tumour development”.
When comparing tumour weights in mice, the authors found “significantly” lower weights in the mGPRC5D and PD1 Ab group than in the control group or each monotherapy group. They also assessed the ability of the vaccine to induce TNFα or IFNγ responses in mouse splenocytes with the ELISPOT assay. Splenocytes from mice that received either mGPRC5D or PD1 Ab exhibited a “significant” increase in the number of spots, and a further increase was observed in the group that had the combination. The combination group had higher frequencies of TNFα+CD8+, IFNγ+CD8+, TNFα +CD4+, and IFNγ+CD4+ T cells in the spleen.
In a flow cytometric analysis of immune cell populations in the spleen, the authors found that treatment with mGPRC5D increased the frequency of CD4+ T cells by over 150% and CD8+ T cells by over 30%. PD1 Ab treatment increased the frequency of both cells by more than 100%. The combination had a “more pronounced effect”; CD4+ T cells increased more than 350% and CD8+ T cells increased by more than 130%. Similar observations were made for DCs, Mϕ, and NK cells in the spleen. For tumour-infiltrating lymphocytes (TILs), the combination approach increased the population of CD8+ and CD4+ T cells, DCs, Mϕ, and NK cells more than the monotherapies.
A human vaccine
As the peptide sequences of mGPRC5D and hGPRC5D are only ~81% identical, a human version of the vaccine is needed. The researchers developed a nanoplasmid construct expressing human GPRC5D (Nano-hGPRC5D). Prophylactic studies found that tumour growth was “significantly suppressed” in the mice group that received Nano-hGPRC5D, which also presented a “marked increase” in serum IgG levels. Other findings include a “significant increase” in the levels of cytokines in the Nano-hGPRC5D group, which suggests a “robust activation of inflammatory cytokines” upon vaccination.
In the spleen and tumours of hGPRC5D-immunised mice, percentages of CD3+, CD4+, and CD8+ T cells and DCs were “significantly increased”. Furthermore, higher frequencies of Th1 secretory cytokine-positive CD3+ T cells were observed in this group. A long-acting protective effect against tumours was implied in “significantly higher percentages” of effector and central memory T cells in the splenocytes of the hGPRC5D group. CD8+ T cells stimulated with the hGPRC5D peptide pool exhibited “superior proliferative ability” compared to the control.
Therapeutic combination
To evaluate the therapeutic efficacy of Nano-hGPRC5D in combination with PD1 Ab, the authors used syngeneic murine models. The combination resulted in “significant tumour regression” compared to either treatment alone. Levels of TNFα, IFNγ, IL-6, IL-12p40, and IL-12p70 increased “significantly” in the combination group, and ELISpot analysis revealed more TNFα- or IFNγ-positive cells in the combination group.
In a flow cytometric analysis of immune cell populations in the spleen and tumour, the combination caused an increase in effector CD8+ and CD4+ T cells, DCs, Mϕ, and NK cells, but a decrease in Treg cells. H&E staining of tumour sections revealed necrotic lesions in the hGPRC5D and combination groups, but the lack of gross histological damage in several major organs supports the safety and clinical potential of the vaccine or combination.
Analysis of the immune cells revealed a “marked increase” in CD3+, CD8+, and CD4+ T cells in the splenic marginal zones of the combination group, consistent with flow cytometry data. There was also an increase in B lymphocytes and follicular DCs in this group. For TILs, the combination therapy also increased the number of CD8+ and CD4+ T cells.
Improving outcomes
Despite therapeutic advancements, high-risk patients with MM “continue to have poor outcomes”, and there are limited agents to prevent MM or progression from MGUS and SMM. The results from the study suggest that PD1 blockade “enhances tumour growth inhibition” in mice treated with the DNA vaccine and highlight the potential of the DNA-based GPRC5D vaccine to “overcome self-tolerance and the prospects of advancing” into clinical trials.
For the latest on cancer vaccine development and combination approaches to disease control, join us at the Congress in Barcelona next week. Don’t forget to subscribe to our weekly newsletters for more vaccine updates.
