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[See attached.] |
CONFIDENTIAL 2.3.2-a-1 |
MRNA WORK PLAN 2023-2025 |
This Work Plan summarizes the activities and deliverables to develop sCAR-T Switch dosing modalities such as, mRNA-based, to deliver switch to subjects who have received CLBR001 switchable CAR-T cells. Calibr, A Division of Scripps Research will carry out the work in conjunction with appropriate CDMOs. |
Work Plan |
Period Covered |
Deliverable |
Background |
Improving dosing regimens is important to reducing burden on patients, increasing patient compliance, and improving the commercial feasibility of the sCAR-T cell platform. As outlined in slides presented to AbbVie leadership on Feb 3rd, 2023, there are multiple options to reduce dosing burden. Of these options, mRNA is... |
Component Activities |
Discovery |
(a) Conduct discovery and feasibility activities for mRNA-liquid nanoparticle (mRNA-LNP) delivery approach |
a. Establish acceptance criteria for preclinical and draft clinical target profile |
i. The initial desired profile of the mRNA-LNP will target a once-monthly dosing, comprising approximately 7 days of exposure of the switch molecule followed by 21 days of no exposure. The target route of administration (ROA) is intravenous |
(IV), although other ROAs may be evaluated. This is similar to the current sCAR19 clinical dosing regimen with SWI019 Fab. Other profiles may be generated and compared. |
b. Identify CDMO for (i) R&D-scale plasmid and mRNA material, (ii) clinical-scale material, and (iii) LNP formulation. The CDMO may be the same or may be different for each requirement. |
(b) Design mRNA for expression of switch. Sequences for the anti-CD19 switch SWI019 or anti-Her2 switch are expected to be used initially for proof of concept. Design will include input from consultants and KOLs. Multiple iterations of design and production are expected to generate a library of sequences including the ... |
a. Determine use of 5' capping technology |
b. Identify appropriate 5' and 3' UTR sequences |
c. Generate codon-optimized mRNA coding sequence |
d. Identify suitable plasmid to harbor components |
e. Identify appropriate signal peptide (leader sequences) |
f. Identify suitable LNP formulations for proof of concept. Multiple LNP formulations may be used to study cell tropism |
(c) Generate LNP-mRNA for R&D scale |
a. Initial CDMO's identified with such capabilities are Trilink, Curia, and Etherna. Other CDMOs may be used, including for the generation of plasmid DNA. |
(d) Characterize LNP-mRNA at the R&D scale. R&D scale will be sufficient for in vitro and in vivo proof of concept studies. |
a. Determine that LNP-mRNA meets established acceptance criteria via analytical testing |
(e) Characterize switch protein production and activity in vitro |
a. Analytical characterization of switch protein production from suitable producer cell line such as K562, including protein production productivity (yields) and protein integrity (mass spec) |
b. Assess functional activity of switch produced by LNP-mRNA in vitro using co-culture assays including CLBR001 and antigen-positive target cells mixed with supernatants from producer cell line transfected with LNP-mRNA. Specific reads will be target cell lysis, cytotoxicity, and upregulation of activation markers on C... |
(f) Assess pharmacokinetics of LNP-mRNA switch in mouse using IV dosing |
a. Conduct pharmacokinetic studies in NSG mice via single and multidose studies and quantitate switch protein in peripheral blood |
b. Calculate the amount of time switch concentrations are over EC50 in plasma for each LNP-mRNA generated. |
c. Other dosing routes of administration may be compared including intranodal for SWI019 or intratumoral for Her2 |
(g) Assess in vivo activity in mouse xenograft models (CD19 positive and/or Her2 positive) |
a. Test the activity of LNP-mRNA switches in NSG mouse xenograft models in combination with CLBR001 switchable CAR-T cells that assess the following variables: |
i. Comparison of multiple LNP-mRNA candidates |
ii. Comparison with Fab-based switches |
b. Success criteria will include reaching target expected exposures and elimination of tumors in mice. |
Lead Optimization & Candidate Selection |
(a) Conduct optimization campaign to identify clinically translatable LNP formulation for a single switch target. |
a. Optimization will be carried out with CDMO and will include consideration for phospholipids, cholesterol, PEGylated lipids, and cationic or ionizable lipids. |
b. Lead opt may also include optimization of 1-methylpseudouridine content of mRNA |
(b) Generate optimized LNP-mRNA candidates |
a. As described above |
(c) Characterize optimized LNP-mRNA candidates at the R&D scale |
a. As described above |
(d) Assess pharmacokinetics of optimized LNP-mRNA candidates in mouse using IV dosing |
a. As described above |
b. In addition, analytical assays for detection of mRNA from LNP-mRNA and/or assays for detecting lipid components will be implemented as necessary. |
(e) Assess in vivo activity of optimized LNP-mRNA candidates |
a. As described above for lead candidates, in addition as described below: |
b. Multiple dose levels of LNP-mRNA |
c. Dose frequencies of LNP-mRNA ranging from monthly to weekly |
d. Tissue PK for mRNA and switch protein will be implemented as appropriate for the target indication. |
(f) Rodent tolerability: Multidose rodent tolerability will be carried out in mouse or rat. The expected dosing frequency of these models are expected to be between weekly and monthly |
a. Assessment of acute phase reactants (e.g., CRP, IL-6, complement, IP-10) will be monitored |
b. Assessment of liver enzyme tests (e.g., ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase) |
c. Tissue tropism of the mRNA may be assessed as appropriate using established methods. |
(g) Non-human primate (NHP) PK and tolerability |
a. Quantitation of switch protein levels in plasma/serum |
b. mRNA from the LNP-mRNA will be tested in serum using quantitative RT-PCR |
c. ADA for anti-PEG and switch antibodies will be assessed |
d. Complement activation factors will be assessed |
e. Liver enzymes will be assessed |
f. Assessment with and without predosing with steroids may be assessed (if warranted based on rodent studies) |
(h) Manufacturing feasibility will include GMP timelines and costs to scale LNP-mRNA to IND |
a. A purification method suitable for clinical development will be used for mRNA |
b. Analytical criteria specific to mRNA to include the following analytics for candidate selection: |
i. Identify (enzymatic degradation) |
ii. RNA integrity (capillary electrophoresis) |
iii. Residual protein (BCA assay) |
iv. Residual DNA (qPCR) |
v. Residual dsRNA (dot blot) |
vi. % cap (LC-MBS) |
vii. Potency (cell-based assay) – optional |
(i) DELIVERABLE: A single candidate will be selected that meets prescribed target profile / development criteria. |
Timeline: approximately 2 year to development candidate is expected. Due to the exploratory nature of the work, the timeline may be adjusted due to results and there is no guarantee that a successful candidate can be generated for an mRNA switch. |
Schedule 2.3.2-b: In-Situ Work Plan |
[See attached.] |
CONFIDENTIAL 2.3.2-b-1 |
IN SITU WORK PLAN 2023-2025 |
This Work Plan summarizes the activities and deliverables to develop an in situ switchable CAR-T platform by Calibr. The in situ platform is expected to be based on an engineered lentiviral vector. Calibr will conduct work to accomplish goals of the program: (a) demonstrate feasibility of in situ approach in the contex... |
Work Plan |
Period Covered |
Deliverable |
Background |
The development of an in situ-based platform for generation of sCAR-T cells in vivo affords many advantages towards the goal of establishing a universal sCAR-T platform. These are outlined in the slide below presented to AbbVie leadership on Feb 3rd, 2023. |
This work plan outlines efforts to create a best-in-class lentiviral vector for in situ delivery of the CAR transgene. The lentiviral platform was selected through a comparison of all available platforms (RNA, retro, transposon, LNP, etc.) based on its low immunogenicity, reduced oncogenic potential (compared with retr... |
dosing. The goal of this model is to (a) set a baseline control for engineering efforts; (b) demonstrate feasibility of activating an in situ-generated sCAR-T cell with a separately administered switch; and (c) test the hypothesis that a low systemic dose of lentiviral vector is sufficient to transduce a small number o... |
1. Non-engineered lentiviral or retroviral vectors will be dosed IV to transduce T cells in situ in fully immunocompetent mice strain |
2. The following parameters will be assessed: |
i. Cell tropism/specificity: On-target activity (sCAR-T cell generation), off-target activity (transduction of cells and tissues other than T cells) |
ii. In vivo sCAR-T cell expansion and phenotyping |
iii. Tolerability (blood chemistry, serum cytokines, weight) |
3. Compare with conventional mouse CAR-T cells targeting CD19 |
a) Engineering of human lentiviral vectors and lead selection: |
1. Establish acceptance criteria for lead vector design for in situ application: |
i. Criteria will be based on cell tropism/selectivity, tolerability, T cell transduction, and sCAR-T cell expansion and antitumor activity as described in the TPP table |
ii. Determine the lowest dose (single dose) that allows for robust sCAR-T expansion through CD19 to later eradicate established solid tumors |
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