Toxicology

The applicant submitted product specific and non-product specific studies; the latter studies were conducted with mRNA vaccine candidates that are based on the same LNP-technology as applied in mRNA-1273.

Repeat dose toxicity

The following seven repeated dose toxicity studies were submitted:

- Study 2308-123: Non-GLP compliant study examining the repeated dose toxicology of mRNA-1273;

- Study 5002045: GLP-compliant study examining the repeated dose toxicology of mRNA-1706, a LNP product containing mRNA that encodes the pre-membrane and envelope structural proteins of Zika virus;

- Study 5002231: GLP-compliant study examining the repeated dose toxicology of mRNA-1706, an LNP product containing mRNA that encodes encoding the prME structural proteins of Zika virus;

- Study 5002033: GLP-compliant study examining the repeated dose toxicology of mRNA-1653, a mRNA vaccine product containing 2 distinct mRNA sequences that encode the full-length membrane-bound fusion proteins of human metapneumovirus and parainfluenza virus type 3;

- Study 5002400: GLP-compliant study examining the repeated dose toxicology of mRNA-1893, a LNP-mRNA vaccine candidate that encodes the prME structural proteins of Zika virus;

- Study 5002034: GLP-compliant study examining the repeated dose toxicology of mRNA-1647, a LNP product containing an equal fraction of 6 mRNAs which encode the full-length cytomegalovirus glycoprotein B (gB) and the pentameric gH/gL/UL128/UL130/UL131A glycoprotein complex;

- Study 5002158: GLP-compliant study examining the repeated dose toxicology of mRNA-1443, a LNP product containing a single mRNA sequence that encodes for a phosphorylation mutant of the CMV phosphoprotein 65 protein.

In all studies the control group was treated with Phosphate-buffered Saline (PBS).

The product-specific Study 2308-123 was not conducted in GLP-compliance, and exhibits major procedural/methodological limitations. In principle these aspects would render this study inadequate for evaluating the repeated dose toxicity of mRNA-1273 to the extent recommended in relevant

are observed between study 2308-123 and the repeated dose toxicity studies conducted with other LNP-mRNA products, the latter studies are considered sufficient to support clinical development and MAA.

The six submitted non-product-specific (but LNP-specific) repeated dose toxicity studies were conducted in GLP-compliance and meet the recommended criteria set out by relevant guidelines.

Considering that the translated antigens of the evaluated mRNA-products are expected to elicit similar immunologic reactions, and considering that all these products are based on the same LNP technology, the extent of the submitted repeated dose toxicity programme is deemed acceptable. In the light of this statement, the GLP and procedural/methodological limitations of study 2308-123 are accepted.

In all studies, at least 2 - 4 doses of the product were applied to male and female Sprague Dawely rats (n = 5 per group and sex in Study 2308-123, n = 10 per group and sex in the other studies) by intramuscular administration, dosing ranged from 9 to 150 μg mRNA/dose. Apart from study 2308-123 in which only in-life endpoints, haematology and binding antibodies were analysed, clinical endpoints, ophthalmology examinations, clinical pathology parameters (haematology, coagulation, and clinical chemistry), post-mortem examinations (necropsy, histo(path)ology), neutralising antibodies and cytokine analysis (usually different interleukins, interferon gamma, acute-phase proteins) were generally assessed in the remaining studies. Reversibility of effects was generally studied after a two weeks recovery period.

In general, the repeated dose toxicology of the tested products proved to be quite similar among the studies, supporting that observed toxicities were not product specific, but rather caused by the immunologic responses towards the translated antigens, and potentially by a contribution of the novel LNP formulation. Test article-related adverse effects were observed at all tested concentrations, dose-dependency was frequently observed.

The following test-article related observations were generally noted in the submitted rat toxicity:

Test article-related in-life observations observed at ≥9 µg/dose included reversible or reversing erythema and oedema at the injection site and transient increase in body temperature at 6 hours post-dose returning to baseline 24 hours post-post-dose.

Haematology changes included increase in white blood cells, neutrophils, and eosinophils and decreased lymphocytes; coagulation changes included increase in fibrinogen and activated partial thromboplastin time; and clinical chemistry changes included decrease in albumin, increase in globulin, and a corresponding decrease in albumin/globulin ratio. Clinical pathology changes generally reversed or were reversing by the end of the 2-week recovery period.

Test article-related transient cytokine increases were observed at ≥9µg/dose at 6 hours post-dose including IFN-γ-induced protein-10, monocyte chemoattractant protein, and macrophage inflammatory protein 1 α. Cytokine changes were generally reversing by the end of the 2-week recovery period.

Post-mortem test article-related and generally dose-dependent changes in organ weights and macroscopic and microscopic findings were observed at ≥9 µg/dose. Organ weight increases were observed in the spleen, liver, and adrenal gland. Organ weight changes were generally reversing by the end of the 2-week recovery period. Macroscopic changes included skin thickening at the injection site and enlarged lymph nodes. Injection site changes completely recovered, and lymph node changes were recovering by the end of the 2-week recovery period. Microscopic changes included mixed cell inflammation at the injection site; increased cellularity and mixed cell inflammation in the inguinal, iliac, and popliteal lymph nodes; decreased cellularity in the splenic periartiolar lymphoid sheath;

increased myeloid cellularity in the bone marrow; and hepatocyte vacuolation and Kupffer cell hypertrophy in the liver. Microscopic changes were generally reversing by the end of the 2-week recovery period.

Genotoxicity

mRNA-1273 contains natural nucleosides and lipid nanoparticles. The applicant submitted genotoxicity data to evaluate the genotoxic potential of the novel excipient SM-102 as well as the final vaccine formulation. The other lipid components contained in the final formulation, i.e. PEG2000-DMG, DSPC and cholesterol, were not separately tested but are contained in the formulation tested in the in vivo genotoxicity studies, which is acceptable. DSPC and cholesterol do not raise any concern in terms of genotoxic potential.

SM-102 was tested for its genotoxic potential in study 9601567 in a bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli. Results did not indicate any evidence of genotoxic activity in this in vitro mutagenicity assay. SM-102 did not show any evidence of genotoxic activity in the conducted in vitro mammalian cell micronucleus test in human peripheral blood lymphocytes (Study 9601567). No cytotoxicity was observed in this assay. Both assays were performed in compliance with GLP.

In in vivo genotoxicity testing, NPI luciferase mRNA in SM-102-containing LNPs was determined to be negative (non-clastogenic) after a single dose of 0.32/6.0, 1.07/20, or 3.21/60 mg/kg NPI luciferase mRNA/SM-102 in Sprague Dawley rats. A statistically significant decrease in PCEs (polychromatic erythrocyte) was observed in the low dose 0.32/6.0 mg/kg NPI luciferase mRNA/SM-102 in the male group only (male and female were tested in separate groups) at the 48-hour time point. This effect did not show dose dependency and after 24 hours was no longer evident.

Increases in cytokines IL-6, MCP-1, MIP-1α, and IP-10 were observed in this study 6 hours after IV administration of 1.07/20 mg/kg and 3.21/60 mg/kg NPI luciferase mRNA/SM-102, respectively.

Reference in this regard is made to the nonclinical pharmacology section, dealing with cytokine release after the intramuscular administration of clinically relevant doses of mRNA-1273 to NHP.

Another GLP-compliant in vivo micronucleus study in rat was performed with mRNA-1706 in SM-102-containing lipid nanoparticles using IV administration. In this study statistically significant increases in micronucleated erythrocytes were reported in both sexes. A strong increase in Molecular initiating event (MIE) was observed 48 hours after the final administration in the highest dose group in male rats (mRNA-1706: 4.0/5.2 mg/kg; SM-102: 54.1 mg/kg). No clear dose-response relationship was reported.

With regards to the positive findings observed in in vivo micronucleus assays, ICH S2(R1) states that in this case ‘all the toxicological data should be evaluated to determine whether a non-genotoxic effect could be the cause or a contributing factor’. In the toxicological studies conducted in rat, various non-genotoxic effects that could impact on the increase of micronucleated erythrocytes in this species were observed: hyperthermia, disturbance of erythropoiesis (lower reticulocyte count, higher red blood cell distribution width) and increase and inflammation of the spleen, which could affect clearance of micronucleated cells from the blood.

Carcinogenicity

No carcinogenicity studies were submitted. This is scientifically acceptable and in line with relevant guidelines on non-clinical development of vaccine candidates. The components of the vaccine formulation are lipids and natural nucleosides that are not expected to have carcinogenic potential.

Reproduction Toxicity

A GLP-compliant reproductive and developmental toxicology (DART) study with mRNA-1273 has been conducted in female Sprague Dawley CD rats.

IM administrations of mRNA-1273 to female SD 1 rats at the human clinical dose, twice before mating and twice during gestation, was associated with non-adverse effects including thin fur cover, swollen hindlimbs and limited usage of the hindlimb. However, there were no mRNA-1273-related effects on female fertility, embryo-foetal or post-natal survival, growth or development in the F1 offspring. The mRNA-1273-related non-adverse effects were limited to an increase in the number of foetuses with common skeletal variations of 1 or more rib nodules and 1 or more wavy ribs, with no effect on the viability and growth on the F1 generation pups.

In this study, no vaccine dose was administered during the early organogenesis, to address the direct embryotoxic effect of the components of the vaccine formulation. However, such a risk is considered low in humans, given the non-live organism nature of mRNA-1273 and the low risk of genotoxic effect of SM-102-containing LNP in humans. The overall pregnancy index was numerically lower in mRNA-1273 vaccinated female rats (84.1%), compared to control animals (93.2%), but remains within the Test Facility’s historical control range (low range being 75%).

Apart from that, no consistent adversities were observed in the male and female reproductive tracts of Sprague Dawley rats during macroscopic and microscopic investigation in the frame of the submitted repeated dose toxicity studies.

Local Tolerance

No stand-alone local tolerance studies were submitted. This is acceptable and in line with relevant guidance on non-clinical vaccine development since local tolerance was evaluated in repeated dose toxicity studies.

In these studies, administration of LNP-mRNA products proved to induce local irritancy and inflammation. These effects can be related to an immunologic response towards the administered mRNA-1273 at and in the vicinity of the injection site, the former being the desired pharmacological mode of action of mRNA-1273. However, the observed local inflammatory response towards LNP-mRNA injection in rats was not only noted in the direct vicinity of the injection site, but also in adjacent tissues and/or organs. For example, subcutaneous tissue, the dermis, epidermis, skeletal muscle (with myofiber degradation), perineurial tissue surrounding the sciatic nerve, and draining lymph nodes in proximity to the injection site were commonly affected by inflammation after LNP-mRNA

administration.