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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 |
2. Design, clone, and generate candidate lentiviral vectors: |
i. Determine T cell specificity and ablation of receptors for other cell types |
ii. Perform optimization of transduction of T cells in peripheral blood (resting T cells) if necessary |
iii. Determine potential for immunogenicity |
3. Conduct biology activities to characterize the lentiviral vectors: |
i. Perform physical and functional titrations |
ii. Perform selectivity assessment in the presence of cell types other than T cells |
iii. Assess sensitivity to human complement and T cell transduction in human whole blood |
4. Conduct in vivo activities for this program: |
i. Profile in vivo vector potency (dose titration), vector biodistribution (if necessary), specificity/cell tropism, and sCART expansion through SWI019 switch |
ii. Determine efficacy in humanized mouse models with implanted solid tumors. In these models, human PBMCs will be engrafted on MHC knock-out mice and transduced in vivo with systemically delivered vectors. sCAR-T cells will be expanded with SWI019 and CD19-positive cells and retargeted against a selected solid tumor t... |
b) Engineering of packaging cell line to improve the vector attributes. |
1. Cell line engineering will attempt to reduce potential for immunogenicity of vectors. Calibr will determine impact of the following approaches: |
i. Knock out B2M and Knock in CD47 genes |
ii. Clone characterization and selection |
iii. Expansion of selected clones |
iv. Lentiviral vector generation employing selected clones |
2. Conduct biology activities to characterize the lentiviral vectors from (b): |
i. Perform physical and functional titrations |
ii. Assess immunogenicity of vectors in the presence of myeloid and other immune cells |
iii. Assess T cell transduction in human whole blood |
3. Conduct in vivo activities in humanized murine models: |
i. Profile of vector potency (dose titration), vector biodistribution/selectivity/cell tropism, and sCAR-T cell expansion through SWI019 switch dosing on similar model as above |
ii. Efficacy in humanized mouse models with implanted solid tumors. In this step, full reconstitution with a human immune system (including T, B, and myeloid cells) will be required to proof the advantages of the engineering strategies implemented. Therefore, humanized models with CD34+ HSC engrafted instead of PBMCs w... |
In situ sCAR-T platform development Discovery Activities |
Surrogate model PoC |
Engineering and generation of human vectors |
In vitro/vivo activities with human vectors |
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