Sprowls CAR T-Cell Therapy

T-lymphocytes, or T-cells, are an integral component of the adaptive immune system that help to _____

Fight off pathogenic invaders and recognize aberrant, unhealthy host cells

T-cells recognize and activate _____ bound peptide fragments

Major histocompatibility complex class I (MHC-I) or class II (MHC-II)

What happens when T-cells activated MHC bound peptide fragments?

Induction of either perforin mediated cell killing (CD8⁺) or recruitment of other immune effector cells (CD4⁺, or T-helper cells) to perpetuate pathogen elimination

Chimeric antigen receptor (CAR) T-cells

Genetically modified T-cell based therapy that is derived from a patient’s own T-cells

• Sometimes cells are collected from the bone marrow after proper differentiation

Since CAR T-cells are derived from a patient’s own T-cells, many of these therapies are _____

Autologous

• Reduces the risk of toxicity or rejection

Following target engagement, CAR T-cells _____

Utilize the same cellular machinery as normal T-cells to induce cell death in cancer cells

• Currently the only FDA approved indication

Is it important to differntiate between CD4 and CD8 when collecting T-cells from a patient?

No

Most common diseases targeted by CAR T-cells

Blood-based cancers

• Multiple myeloma, lymphoblastic leukemia, and varying lymphomas

Under normal circumstances, T-cells engage MHC-bound antigens on either professional antigen presenting cells or any nucleated cell in the body, causing _____

Immune activation against exogenous or endogenous pathogens, respectively

Binding of a T-cell to an MHC-bound antigen, in conjunction with other co-stimulatory elements and adhesion molecule pairings creates _____

An immunological synapse → leads to activation of the T-cell and ideally subsequent pathogen elimination

Unlike our T-cells, CAR T-cells undergo _____ activation

MHC-independent

• Still require co-stimulation and adhesion to the target cell

CAR T-cells do not engage with target cells through the TCR, but through _____

A genetically engineered element of an antibody called a single-chain variable fragment (scFv)

scFv elements have very high specificity for _____

Specific epitopes of extracellularly expressed protein antigens that promote cancer cell growth and survival

• The most common of which are currently either CD19 or B-cell maturation antigen (BCMA)

Since CARs have high specificity, they will only engage with cells that express a specific antigen. Does this mean there are no toxicities related to these treatments?

No

For a T-cell to express the CAR, the genetic material must be _____

Added to the cell’s existing genome

Adding genetic material to a cell requies that some molecular technology must be used to incorporate the right genetic material into the host cell DNA without damaging the cell or inducing apoptosis. This is accomplished using _____

Viral vectors

All approved CAR T-cell therapies use _____ vectors

Retroviral-based

_____ vectors are used in Yescarta and Tecartus

𝛾-retroviral

_____ vectors are used in Kymriah, Breyanzi, Abecma, Carvykti, and Aucatzyl

Lentiviral

Retroviral-based vectors are used for CAR T-cell therapies because of their ability to:

Efficiently package genetic material cargo (~8-10 kb)

Low immunogenicity

Capacity for integration of DNA into host genome

Producer cell lines are used in vitro to create _____

A stable viral particle that will deliver the target gene to the host T-cell for transduction, and subsequent expression of the CAR

Nearly all retroviral vectors are prepared using the same gene elements:

gag

pol

env

Transgene

gag

Encodes for the viral cpasid and structural components of the virus

pol

Encodes for enzymes required to synthesize DNA to be added to the host cell (reverse transcriptase and integrase)

env

Encodes for the envelope portion of the virus and allows for precise transfection of patient’s T-cells

Transgene

Encodes for the actual CAR gene itself and the promoter region to express it

T-cells are collected from the patient through a process called _____

Leukapheresis

What occurs in leukapheresis?

A patient’s blood is collected, the WBCs are extracted, and the remaining blood is returned to the patient

The resulting WBCs are centrifuged and separated to ensure only T-cells are collected

Centrifugation separated mononuclear cells based on _____

Size with the help of a density gradient

After leukapheresis and centrifugation, the resulting cell population still contains multiple cell types and must be further purified to obtain only T-cells. This can be done through _____

Magnetic associated cell sorting (MACS) or flow cytometry and fluorescence activated cell sorting (FACS)

MACS and FACS utilize _____ to ensure only T-cells are collected

Surface expressed CD3/CD28

Once T-cells are engineered to express the CAR, they are allowed to _____

Divide and expand in a bioreactor to obtain a high enough number to be formulated and delivered to the patient

The final formulation of CAR T-cells is _____

Cryopreserved until the patient is ready to be infused with their personalized CAR T-cell therapy

Before the patient can be infused with CAR T-cell therapy, they must receive _____

A short course of immunodepleting chemotherapy

• Fluderabine or cyclophosphomide

Purpose of immunodepleting chemotherapy before CAR T-cell infusion

Allows for maximizing of CAR T-cell expansion within the patient

Creates an environment with increased efficacy

What are the most common side effects related to CAR T-cell therapy?

Cytokine Release Syndrome (CRS)

Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS)

Cytokine Release Syndrome (CRS)

Caused by the extreme activation of other components of the immune system

Upon recognition of the target tumor cells, CAR T-cells will release massive amounts of cytokines (TNF-alpha, IFN-gamma) to _____

Propagate an immune response against the tumor cell and initiate increased perforin dependent cell death of tumor cells

• Results in the release of damage associated molecular patterns (DAMPs)

DAMPs further amplify the immune response through _____

Binding to other effector immune cells such as macrophages, which in turn release more inflammatory cytokines (TFN-alpha, IFN-gamma, IL-6, IL-2, IL1-beta, etc.)

Symptoms of CRS

Range in presentation

Begin with low-grade fever, myalgia, joint pain, and general malaise

Can progress rapidly to hypotension, hypoxia, and tachycardia

Ultimately manifest as cardiorespiratory dysfunction and multiple organ system failure

ICANS nearly always occurs _____

With a peak several days after CRS

• Rare cases of independent ICANS do exist

Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS)

Excessive release of cytokines (particularly TNF-alpha, IL-6, and IL1-beta) can cause activation of endothelial cells and subsequent disruption of the Ang-TIE2 axis, promoting instability and diminished integrity of the BBB

This cascade of events results in endothelial cell separation/death, capillary leak, and exposure of the CNS to cytokines and other components of the immune system

Expxosure of astrocytes to cytokines leads to _____

Reactive astrogliosis

• Known to propagate cerebral edema

• Astrocytes can release VEGF-A and MMPs, which function to increase vascular permeability

Microglia are brain resident macrophages that can become acitvated during cytokine penetration beyond the BBB. Once activated they _____

Continue to propagate an intracranial immune response through release of proinflammatory cytokines

What is the ultimate result of ICANS, and what symptoms does it present with?

Results in cytokine-induced neuronal injury and increased excitotoxicity

Presents with symptoms of headaches, neuro-motor issues, and speech difficulties

Progresses to severe symptoms such as seizures and cerebral edema

What is the target of Kymriah, Yescarta, Tecartus, Breyanzi, and Aucatzyl?

CD19

What is the target of Abecma and Carvykti?

BCMA

How are CAR T-cell therapies dosed?

Infusion based on a number of CAR-positive viable cells per kg of body weight

CAR T-cell therapies should be infused within _____ after thawing

30 minutes

• Ensures cell viability and functionality

• Limits the effects of cryopreservatives on CAR T-cells

• Prevents aggregation